Methods for genetic control of insect infestations in plants and compositions thereof

ABSTRACT

The present invention relates to control of pest infestation by inhibiting one or more biological functions. The invention provides methods and compositions for such control. By feeding one or more recombinant double stranded RNA molecules provided by the invention to the pest, a reduction in pest infestation is obtained through suppression of gene expression. The invention is also directed to methods for making transgenic plants that express the double stranded RNA molecules, and to particular combinations of transgenic pesticidal agents for use in protecting plants from pest infestation.

PRIORITY CLAIM

This application claims the priority of U.S. Provisional ApplicationSer. No. 60/718,034, filed Sep. 16, 2005, which application isincorporated herein by reference in its entirety.

The Sequence Listing is submitted on one compact disc (Copy 1), togetherwith a duplicate thereof (Copy 2), each created on Sep. 15, 2006, andeach containing one 669 kb file entitled “MNDE002.APP.TXT.” The materialcontained on the compact disc is specifically incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to genetic control of pestinfestations. More specifically, the present invention relates torecombinant DNA technologies for post-transcriptionally repressing orinhibiting expression of target coding sequences in the cell of a pestto provide a pest-protective effect.

2. Description of Related Art

The environment in which humans live is replete with pest infestation.Pests including insects, arachnids, crustaceans, fungi, bacteria,viruses, nematodes, flatworms, roundworms, pinworms, hookworms,tapeworms, trypanosomes, schistosomes, botflies, fleas, ticks, mites,and lice and the like are pervasive in the human environment. Amultitude of means have been utilized for attempting to controlinfestations by these pests. Compositions for controlling infestationsby microscopic pests such as bacteria, fungi, and viruses have beenprovided in the form of antibiotic compositions, antiviral compositions,and antifungal compositions. Compositions for controlling infestationsby larger pests such as nematodes, flatworm, roundworms, pinworms,heartworms, tapeworms, trypanosomes, schistosomes, and the like havetypically been in the form of chemical compositions that can be appliedto surfaces on which pests are present or administered to infestedanimals in the form of pellets, powders, tablets, pastes, or capsulesand the like. There is a great need in the art for improvement of thesemethods and particularly for methods that would benefit the environmentrelative to the prior techniques.

Commercial crops are often the targets of insect attack. Substantialprogress has been made in the last a few decades towards developing moreefficient methods and compositions for controlling insect infestationsin plants. Chemical pesticides have been very effective in eradicatingpest infestations. However, there are several disadvantages to usingchemical pesticidal agents. Chemical pesticidal agents are notselective. Applications of chemical pesticides intended to controlinvertebrate pests, such as coleopteran insects including corn rootwormspecies that are harmful to various crops and other plants, exert theireffects on non-target fauna as well, often effectively sterilizing afield for a period of time over which the pesticidal agents have beenapplied. Chemical pesticidal agents persist in the environment andgenerally are slow to be metabolized, if at all. They accumulate in thefood chain, and particularly in the higher predator species.Accumulations of these chemical pesticidal agents results in thedevelopment of resistance to the agents and in species higher up theevolutionary ladder, can act as mutagens and/or carcinogens to causeirreversible and deleterious genetic modifications. Thus there has beena particularly long felt need for environmentally friendly methods forcontrolling or eradicating insect infestation on or in plants, i.e.,methods that are selective, environmentally inert, non-persistent, andbiodegradable, and that fit well into pest resistance managementschemes.

Compositions that include Bacillus thuringiensis (Bt) bacteria have beencommercially available and used as environmentally safe and acceptableinsecticides for more than thirty years. The insecticidal effect of Btbacteria do not persist in the environment, are highly selective as tothe target species affected, exert their effects only upon ingestion bya target pest, and have been shown to be harmless to plants and othernon-targeted organisms, including humans. Transgenic plants containingone or more genes encoding insecticidal Bt protein are also available inthe art and are remarkably efficient in controlling insect pestinfestation. A substantial result of the use of recombinant plantsexpressing Bt insecticidal proteins is a marked decrease in the amountof chemical pesticidal agents that are applied to the environment tocontrol pest infestation in crop fields in areas in which suchtransgenic crops are used. The decrease in application of chemicalpesticidal agents has resulted in cleaner soils and cleaner watersrunning off of the soils into the surrounding streams, rivers, ponds andlakes. In addition to these environmental benefits, there has been anoticeable increase in the numbers of beneficial insects in crop fieldsin which transgenic insect resistant crops are grown because of thedecrease in the use of chemical insecticidal agents.

Antisense methods and compositions have been reported in the art and arebelieved to exert their effects through the synthesis of asingle-stranded RNA molecule that in theory hybridizes in vivo to asubstantially complementary sense strand RNA molecule. Antisensetechnology has been difficult to employ in many systems for threeprinciple reasons. First, the antisense sequence expressed in thetransformed cell is unstable. Second, the instability of the antisensesequence expressed in the transformed cell concomitantly createsdifficulty in delivery of the sequence to a host, cell type, orbiological system remote from the transgenic cell. Third, thedifficulties encountered with instability and delivery of the antisensesequence create difficulties in attempting to provide a dose within therecombinant cell expressing the antisense sequence that can effectivelymodulate the level of expression of the target sense nucleotidesequence.

There have been few improvements in technologies for modulating thelevel of gene expression within a cell, tissue, or organism, and inparticular, a lack of developed technologies for delaying, repressing orotherwise reducing the expression of specific genes using recombinantDNA technology. Furthermore, as a consequence of the unpredictability ofthese approaches, no commercially viable means for modulating the levelof expression of a specific gene in a eukaryotic or prokaryotic organismis available.

Double stranded RNA mediated inhibition of specific genes in variouspests has been previously demonstrated. dsRNA mediated approaches togenetic control have been tested in the fruit fly Drosophilamelanogaster (Kennerdell and Carthew, 1998; Kennerdell and Carthew,2000). Kennerdell and Carthew (1998) describe a method for delivery ofdsRNA involved generating transgenic insects that express doublestranded RNA molecules or injecting dsRNA solutions into the insect bodyor within the egg sac prior to or during embryonic development.

Research investigators have previously demonstrated that double strandedRNA mediated gene suppression can be achieved in nematodes either byfeeding or by soaking the nematodes in solutions containing doublestranded or small interfering RNA molecules and by injection of thedsRNA molecules. Rajagopal et. al. (2002) described failed attempts tosuppress an endogenous gene in larvae of the insect pest Spodopteralitura by feeding or by soaking neonate larvae in solutions containingdsRNA specific for the target gene, but were successful in suppressionafter larvae were injected with dsRNA into the hemolymph of 5^(th)instar larvae using a microapplicator. Recently, Yadav et al. (2006)reported that host-generated dsRNA produced in a plant can protect suchplants from infection by nematodes. Similarly, U.S. Patent App. Pub. No.2003/0150017 prophetically described a preferred locus for inhibition ofthe lepidopteran larvae Helicoverpa armigera using dsRNA delivered tothe larvae by ingestion of a plant transformed to produce the dsRNA. WO2005/110068 teaches providing, in the diet of corn rootworm (CRW),CRW-specific dsRNA directed to essential CRW genes. The dsRNA isprovided in the CRW diet in-vitro and in-planta, with the result thatCRW larvae are stunted or killed after feeding on the diet, and thiseffect was demonstrated for several different genes.

Therefore, there has existed a need for identifying efficaciousnucleotide sequences for use in improved methods of modulating geneexpression by repressing, delaying or otherwise reducing gene expressionwithin a particular coleopteran pest for the purpose of controlling pestinfestation or to introduce novel phenotypic traits.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of inhibiting expressionof a target gene in a coleopteran pest. In certain embodiments, themethod comprises modulating or inhibiting expression of one or moretarget genes in a coleopteran pest that causes cessation of feeding,growth, development, reproduction and/or infectivity and eventuallyresult in the death of the insect. The method comprises introduction ofpartial or fully, stabilized double-stranded RNA (dsRNA), including itsmodified forms such as small interfering RNA (siRNA) sequences, into thecells or into the extracellular environment, such as the midgut, withina coleopteran pest body wherein the dsRNA enters the cells and inhibitsexpression of at least one or more target genes and wherein theinhibition exerts a deleterious effect upon the coleopteran pest. Themethods and associated compositions may be used for limiting oreliminating coleopteran pest infestation in or on any pest host, pestsymbiont, or environment in which a pest is present by providing one ormore compositions comprising the dsRNA molecules described herein in thediet of the pest. The method will find particular benefit for protectingplants from insect attack. In one embodiment, the pest is defined ascomprising a digestive system pH within the range of from about 4.5 toabout 9.5, from about 5 to about 9, from about 6 to about 8, and fromabout pH 7.0.

In another aspect, the present invention provides exemplary nucleic acidcompositions that are homologous to at least a portion of one or morenative nucleic acid sequences in a target pest. In certain embodiments,the pest is selected from among Diabrotica sp. including Western CornRootworm (WCR, Diabrotica virgifera or Diabrotica virgifera virgifera),Southern Corn Rootworm (SCR, Diabrotica undecimpunctata howardi),Mexican Corn Rootworm (MCR, Diabrotica virgifera zea), Brazilian CornRootworm (BZR, Diabrotica balteata, Diabrotica viridula, Diabroticaspeciosa), Northern Corn rootworm (NCR, Diabrotica barberi), Diabroticaundecimpunctata; as well as Colorado Potato Beetle (CPB, Leptinotarsadecemlineata), Red Flour Beetle (RFB, Tribolium castaneum), and MexicanBean Beetle (Epilachna varivestis). In other embodiments the pest isselected from among Lepidopteran insects including European Corn Borer(ECB, Ostrinia nubilalis), Black Cutworm (BCW, Agrotis ipsilon), CornEarworm (CEW, Helicoverpa zea), Fall Armyworm (FAW, Spodopterafrugiperda), Cotton Ball Weevil (BWV, Anthonomus grandis), silkworms(Bombyx mori) and Manduca sexta, and from Dipteran insects includingDrosophila melanogaster, Anopheles gambiae, and Aedes aegypti. Specificexamples of such nucleic acids provided by the invention are given inthe attached sequence listing as SEQ ID NO:1 through SEQ ID NO:906.

In yet another aspect, the invention provides a method for suppressionof gene expression in a coleopteran pest such as a corn rootworm orrelated species that comprises the step of providing in the diet of thepest a gene suppressive amount of at least one dsRNA moleculetranscribed from a nucleotide sequence as described herein, at least onesegment of which is complementary to an mRNA sequence within the cellsof the pest. The method may further comprise observing the death,inhibition, stunting, or cessation of feeding of the pest. A dsRNAmolecule, including its modified form such as an siRNA molecule, fed toa pest in accordance with the invention may be at least from about 80,81, 82, 83, 84, 85, 86, 87, 88 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or about 100% identical to a RNA molecule transcribed from anucleotide sequence selected from the group consisting of SEQ ID NO:1through SEQ ID NO:906. In particular embodiments, the nucleotidesequence may be selected from the group consisting of SEQ ID NO:697, SEQID NOs:813-819, SEQ ID NO:841, and SEQ ID NO:874.

Accordingly, in another aspect of the present invention, a set ofisolated and purified nucleotide sequences as set forth in SEQ ID NO:1through SEQ ID NO:906 is provided. The present invention provides astabilized dsRNA molecule or the expression of one or more miRNAs forinhibition of expression of a target gene in a coleopteran pestexpressed from these sequences and fragments thereof. A stabilizeddsRNA, including a miRNA or siRNA molecule can comprise at least twocoding sequences that are arranged in a sense and an antisenseorientation relative to at least one promoter, wherein the nucleotidesequence that comprises a sense strand and an antisense strand arelinked or connected by a spacer sequence of at least from about five toabout one thousand nucleotides, wherein the sense strand and theantisense strand may be a different length, and wherein each of the twocoding sequences shares at least 80% sequence identity, at least 90%, atleast 95%, at least 98%, or 100% sequence identity, to any one or morenucleotide sequence(s) set forth in set forth in SEQ ID NO: 1 throughSEQ ID NO:906.

Further provided by the invention is a fragment or concatemer of anucleic acid sequence selected from the group consisting of SEQ ID NO:1through SEQ ID NO:906. In particular embodiments, the nucleotidesequence may comprise a fragment or concatemer of a sequence selectedfrom the group consisting of SEQ ID NO:697, SEQ ID NOs:813-819, SEQ IDNO:841, and SEQ ID NO:874.

The fragment may be defined as causing a the death, inhibition,stunting, or cessation of feeding of a pest when expressed as a dsRNAand provided to the pest. The fragment may, for example, comprise atleast about 19, 21, 23, 25, 40, 60, 80, 100, 125 or more contiguousnucleotides of any one or more of the sequences in SEQ ID NO:1 throughSEQ ID NO:906, or a complement thereof. One beneficial DNA segment foruse in the present invention is at least from about 19 to about 23, orabout 23 to about 100 nucleotides up to about 2000 nucleotides or morein length. Particularly useful will be dsRNA sequences including about23 to about 300 nucleotides homologous to a pest target sequence. Theinvention also provides a ribonucleic acid expressed from any of suchsequences including a dsRNA. A sequence selected for use in expressionof a gene suppression agent can be constructed from a single sequencederived from one or more target pests and intended for use in expressionof an RNA that functions in the suppression of a single gene or genefamily in the one or more target pests, or that the DNA sequence can beconstructed as a chimera from a plurality of DNA sequences.

In yet another aspect, the invention provides recombinant DNA constructscomprising a nucleic acid molecule encoding a dsRNA molecule describedherein. The dsRNA may be formed by transcription of one strand of thedsRNA molecule from a nucleotide sequence which is at least from about80% to about 100% identical to a nucleotide sequence selected from thegroup consisting of SEQ ID NO:1 through SEQ ID NO:906. Such recombinantDNA constructs may be defined as producing dsRNA molecules capable ofinhibiting the expression of endogenous target gene(s) in a pest cellupon ingestion. The construct may comprise a nucleotide sequence of theinvention operably linked to a promoter sequence that functions in thehost cell. Such a promoter may be tissue-specific and may, for example,be specific to a tissue type which is the subject of pest attack. In thecase of rootworms, for example, it may be desired to use a promoterproviding root-preferred expression.

Nucleic acid constructs in accordance with the invention may comprise atleast one non-naturally occurring nucleotide sequence that can betranscribed into a single stranded RNA capable of forming a dsRNAmolecule in vivo through hybridization. Such dsRNA sequences selfassemble and can be provided in the diet of a coleopteran pest toachieve the desired inhibition.

A recombinant DNA construct may comprise two different non-naturallyoccurring sequences which, when expressed in vivo as dsRNA sequences andprovided in the diet of a coleopteran pest, inhibit the expression of atleast two different target genes in the cell of the coleopteran pest. Incertain embodiments, at least 3, 4, 5, 6, 8 or 10 or more differentdsRNAs are produced in a cell or plant comprising the cell that have apest-inhibitory effect. The dsRNAs may expressed from multipleconstructs introduced in different transformation events or could beintroduced on a single nucleic acid molecule. The dsRNAs may beexpressed using a single promoter or multiple promoters. In oneembodiments of the invention, single dsRNAs are produced that comprisenucleic acids homologous to multiple loci within a pest.

In still yet another aspect, the invention provides a recombinant hostcell having in its genome at least one recombinant DNA sequence that istranscribed to produce at least one dsRNA molecule that functions wheningested by a coleopteran pest to inhibit the expression of a targetgene in the pest. The dsRNA molecule may be encoded by any of thenucleic acids described herein and as set forth in the sequence listing.The present invention also provides a transformed plant cell having inits genome at least one recombinant DNA sequence described herein.Transgenic plants comprising such a transformed plant cell are alsoprovided, including progeny plants of any generation, seeds, and plantproducts, each comprising the recombinant DNA.

The methods and compositions of the present invention may be applied toany monocot and dicot plant, depending on the coleopteran pest controldesired. Specifically, the plants are intended to include, withoutlimitation, alfalfa, aneth, apple, apricot, artichoke, arugula,asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry,broccoli, brussel sprouts, cabbage, canola, cantaloupe, carrot, cassava,cauliflower, celery, cherry, cilantro, citrus, clementine, coffee, corn,cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus,fennel, figs, gourd, grape, grapefruit, honey dew, jicama, kiwifruit,lettuce, leeks, lemon, lime, Loblolly pine, mango, melon, mushroom, nut,oat, okra, onion, orange, an ornamental plant, papaya, parsley, pea,peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum,pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio,radish, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach,squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato,sweetgum, tangerine, tea, tobacco, tomato, turf, a vine, watermelon,wheat, yams, and zucchini plants. Thus, a plant transformed with arecombinant DNA sequence as set forth in SEQ ID NO: 1 through SEQ IDNO:906, or concatemer, fragment, or complement thereof, that istranscribed to produce at least one dsRNA molecule that functions wheningested by a coleopteran pest to inhibit the expression of a targetgene in the pest is also provided by the invention. In particularembodiments, the recombinant DNA sequence may be selected from the groupconsisting of SEQ ID NO:697, SEQ ID NOs:813-819, SEQ ID NO:841, and SEQID NO:874, or fragment, complement, or concatemer thereof.

The invention also provides combinations of methods and compositions forcontrolling coleopteran pest infestations. One means provides a dsRNAmethod as described herein for protecting plants from insect infestationalong with one or more insecticidal agents that exhibit featuresdifferent from those exhibited by the dsRNA methods and compositions.For example, one or more Bt proteins may be provided in the diet ofinsect pests in combination with one or more dsRNAs as described herein.A composition formulated for topical application or derived using atransgenic approach that combines dsRNA methods and compositions with Btmay be used to provide synergies that were not known previously in theart for controlling insect infestation. One synergy is the reduction inthe level of expression required for either the dsRNA(s) or the Btprotein(s). When combined together, a lower effective dose of each pestcontrol agent could be used. It is believed that the Bt insecticidalproteins create entry pores through which the dsRNA molecules are ableto penetrate more effectively into spaces remote from the gut of theinsect pest, or more efficiently into the cells in the proximity oflesions created by the Bt proteins, thus requiring less of either the Btor the dsRNA to achieve the desired insecticidal result or the desiredinhibition or suppression of a targeted biological function in thetarget pest.

The present invention therefore provides a composition that contains twoor more different pesticidal agents each toxic to the same pest orinsect species, at least one of which comprises a dsRNA describedherein. In certain embodiments, the second agent can be an agentselected from the group consisting of a patatin, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, a Bacillus sphaericus insecticidal protein, and alignin. A Bacillus thuringiensis insecticidal protein can be any of anumber of insecticidal proteins including but not limited to a Cry1, aCry3, a TIC851, a CryET70, a Cry22, a TIC901, a TIC1201, a TIC407, aTIC417, a binary insecticidal protein CryET33 and CryET34, a binaryinsecticidal protein CryET80 and CryET76, a binary insecticidal proteinTIC100 and TIC101, a binary insecticidal protein PS149B1, a VIPinsecticidal protein, a TIC900 or related protein, or combinations ofthe insecticidal proteins ET29 or ET37 with insecticidal proteins TIC810or TIC812, and insecticidal chimeras of any of the precedinginsecticidal proteins.

A ribonucleic acid that is provided in a diet can be provided in anartificial diet formulated to meet particular nutritional requirementsfor maintaining a pest on such diet. The diet may be supplemented with apest controlling amount of an RNA that has been purified from a separateexpression system to determine a pest controlling amount of RNAcomposition or to determine extent of suppressive activity uponingestion of the supplemented diet by the pest. The diet can also be arecombinant cell transformed with a DNA sequence constructed forexpression of the agent, the RNA, or the gene suppression agent. Uponingestion of one or more such transformed cells by the pest, a desiredphenotypic result is observed, indicating that the agent has functionedto inhibit the expression of a target nucleotide sequence that is withinthe cells of the pest.

A gene targeted for suppression can encode an essential protein, thepredicted function of which is selected from the group consisting ofmuscle formation, juvenile hormone formation, juvenile hormoneregulation, ion regulation and transport, protein synthesis andtransport, digestive enzyme synthesis, maintenance of cell membranepotential, amino acid biosynthesis, amino acid degradation, spermformation, pheromone synthesis, pheromone sensing, antennae formation,wing formation, leg formation, development and differentiation, eggformation, larval maturation, digestive enzyme formation, haemolymphsynthesis, haemolymph maintenance, neurotransmission, cell division,energy metabolism, respiration, an unknown function, and apoptosis.

Another aspect of the present invention also provides methods forimproving the yield of a crop produced from a crop plant subjected toinsect pest infestation, said method comprising the steps of a)introducing a polynucleotide comprising a sequence selected from SEQ IDNO:1 through SEQ ID NO:906 or a complement or concatemer or fragmentthereof into said crop plant; and b) cultivating the crop plant to allowthe expression of said polynucleotide, wherein expression of thepolynucleotide inhibits feeding by insect pests and loss of yield due topest infestation.

In certain embodiments, the expression of the polynucleotide produces anRNA molecule that suppresses at least a first target gene in an insectpest that has ingested a portion of said crop plant, wherein the targetgene performs at least one essential function selected from the groupconsisting of feeding by the pest, viability of the pest, pest cellapoptosis, differentiation and development of the pest or any pest cell,sexual reproduction by the pest, muscle formation, muscle twitching,muscle contraction, juvenile hormone formation and/or reduction,juvenile hormone regulation, ion regulation and transport, maintenanceof cell membrane potential, amino acid biosynthesis, amino aciddegradation, sperm formation, pheromone synthesis, pheromone sensing,antennae formation, wing formation, leg formation, egg formation, larvalmaturation, digestive enzyme formation, haemolymph synthesis, haemolymphmaintenance, neurotransmission, larval stage transition, pupation,emergence from pupation, cell division, energy metabolism, respiration,cytoskeletal structure synthesis and maintenance, nucleotide metabolism,nitrogen metabolism, water use, water retention, and sensory perception.

In other embodiments, the insect pest is a corn rootworm pest selectedfrom the group consisting of Diabrotica undecimpunctata howardi(Southern Corn Rootworm (SCR)), Diabrotica virgifera virgifera (WesternCorn Rootworm (WCR)), Diabrotica barberi (Northern Corn Rootworm (NCR)),Diabrotica virgifera zea (Mexican Corn Rootworm (MCR)), Diabroticabalteata (Brazilian Corn Rootworm (BZR)), Diabrotica viridula (BrazilianCorn Rootworm (BZR)), and Diabrotica speciosa (Brazilian Corn Rootworm(BZR)).

Methods for improving the drought tolerance of a crop produced from acrop plant subjected to insect pest infestation, said method comprisingthe steps of a) introducing a polynucleotide sequence selected from SEQID NO:1 through SEQ ID NO:906, or a fragment thereof, into said cropplant; and b) cultivating the crop plant to allow the expression of saidpolynucleotide, wherein expression of the polynucleotide inhibitsfeeding by insects pests and loss of drought tolerance due to pestinfestation, are also provided.

Yet another aspect of the invention further provides agronomically andcommercially important products and/or compositions of matter including,but not limited to, animal feed, commodities, products and by-productsthat are intended for use as food for human consumption or for use incompositions and commodities that are intended for human consumptionincluding but not limited to corn flour, corn meal, corn syrup, cornoil, corn starch, popcorn, corn cakes, cereals, and the like. Suchcompositions may be defined as containing detectable amounts of anucleotide sequence set forth herein, and thus are also diagnostic forany transgenic event containing such nucleotide sequences. Theseproducts are useful at least because they are likely to be derived fromcrops propagated with fewer pesticides and organophosphates as a resultof their incorporation of the nucleotides of the present invention forcontrolling the infestation of coleopteran pests in plants. Suchcommodities and commodity products can be produced from seed producedfrom a transgenic plant, wherein the transgenic plant expresses RNA fromone or more contiguous nucleotides of the present invention ornucleotides of one or more coleopteran pests and the complementsthereof. Such commodities and commodity products may also be useful incontrolling coleopteran pests of such commodity and commodity products,such as for example, control of flour weevils, because of the presencein the commodity or commodity product of the pest gene suppressive RNAexpressed from a gene sequence as set forth in the present invention.

A method of producing such a commodity product comprising obtaining aplant transformed with a polynucleotide comprising a sequence selectedfrom the group consisting of SEQ ID NO:1 through SEQ ID NO:906, or aconcatemer or fragment or complement thereof, and preparing a commodityproduct from the plant or part thereof is also provided. Further, amethod of producing food or feed, comprising obtaining a planttransformed with a polynucleotide selected from the group consisting ofSEQ ID NO:1 through SEQ ID NO:906 or a fragment or complement thereof,and preparing food or feed from said plant or part thereof is yetanother aspect of the invention.

The invention also provides a computer readable medium having recordedthereon one or more of the nucleotide sequences as set forth in SEQ IDNO:1 through SEQ ID NO:906, or complements thereof, for use in a numberof computer based applications, including but not limited to DNAidentity and similarity searching, protein identity and similaritysearching, transcription profiling characterizations, comparisonsbetween genomes, and artificial hybridization analyses.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Bioassay of F1 corn plant events transformed with pMON98503 (SEQID NO:820) and challenged with Western Corn Rootworm (WCR).

FIG. 2: Bioassay of F1 corn plant events transformed with pMON98504comprising concatemer C1 (SEQ ID NO:821) and challenged with WesternCorn Rootworm (WCR).

FIG. 3: Selection of Dv49 and Dv248 fragments and schematic design ofDv49-Dv248 concatemer C38.

FIG. 4: dsRNAs F1-F13 synthesized based on concatemer C38.

FIG. 5: DV49-DV248 concatemer 38 dose response (Fragments F1-F6).

FIG. 6: DV49-DV248 concatemer 38 dose response (Fragments F7-F10).

FIG. 7: DV49-DV248 concatemer 38 dose response (Fragments F11-F13).

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention.

The present invention provides methods and compositions for geneticcontrol of pest infestations. For example, the present inventionprovides recombinant DNA technologies to post-transcriptionally repressor inhibit expression of a target coding sequence in the cell of a pestto provide a pest-protective effect by feeding to the pest one or moredouble stranded or small interfering ribonucleic acid (RNA) moleculestranscribed from all or a portion of a target coding sequence, therebycontrolling the infestation. Therefore, the present invention relates tosequence-specific inhibition of expression of coding sequences usingdouble-stranded RNA (dsRNA), including small interfering RNA (siRNA), toachieve the intended levels of pest control.

Isolated and substantially purified nucleic acid molecules including butnot limited to non-naturally occurring nucleotide sequences andrecombinant DNA constructs for transcribing dsRNA molecules of thepresent invention are provided that suppress or inhibit the expressionof an endogenous coding sequence or a target coding sequence in the pestwhen introduced thereto. Transgenic plants that (a) contain nucleotidesequences encoding the isolated and substantially purified nucleic acidmolecules and the non-naturally occurring recombinant DNA constructs fortranscribing the dsRNA molecules for controlling plant pestinfestations, and (b) display resistance and/or enhanced tolerance tothe insect infestations, are also provided. Compositions containing thedsRNA nucleotide sequences of the present invention for use in topicalapplications onto plants or onto animals or into the environment of ananimal to achieve the elimination or reduction of pest infestation arealso described.

The inventors have herein discovered that, contrary to the teachings inthe prior art, feeding a composition containing double stranded RNAmolecules consisting of sequences found within one or more expressednucleotide sequences of a coleopteran species to the species from whichthe nucleotide sequences were obtained results in the inhibition of oneor more biological functions within the coleopteran species.Particularly, the inventors have discovered that feeding the doublestranded RNA molecules described herein to crop pest species such ascorn rootworms results in the death or inhibition of development anddifferentiation of insect pests that ingest these compositions.

The inventors have identified the nucleotide sequences described hereinas providing plant protective effects against coleopteran pest species.Amino acid sequences encoded by the cDNA sequences have been deduced andcompared to known amino acid sequences. Many of the sequences arepredicted to encode proteins that have some annotation informationassociated with them. The annotation information that is associated witha particular nucleotide sequence and protein sequence encoded therefromis based on homology or similarity between the amino acid sequencesdeduced through translation of the coding sequences described herein asset forth and amino acid sequences that are known in the art in publiclyavailable databases.

cDNA sequences encoding proteins or parts of proteins essential forsurvival, such as amino acid sequences involved in various metabolic orcatabolic biochemical pathways, cell division, reproduction, energymetabolism, digestion, neurological function and the like were selectedfor use in preparing double stranded RNA molecules that were provided inthe diet of coleopteran pests. As described herein, ingestion by atarget pest of compositions containing one or more dsRNAs, at least onesegment of which corresponds to at least a substantially identicalsegment of RNA produced in the cells of the target pest, resulted indeath, stunting, or other inhibition of the target pest. These resultsindicated that a nucleotide sequence, either DNA or RNA, derived from acoleopteran pest can be used to construct plant cells resistant toinfestation by the pest. The pest host, for example, can be transformedto contain one or more of the nucleotide sequences derived from thecoleopteran pest. The nucleotide sequence transformed into the pest hostor symbiont may encode one or more RNAs that form into a dsRNA sequencein the cells or biological fluids within the transformed host orsymbiont, thus making the dsRNA available in the diet of the pestif/when the pest feeds upon the transgenic host or symbiont, resultingin the suppression of expression of one or more genes in the cells ofthe pest and ultimately the death, stunting, or other inhibition of thepest

The present invention relates generally to genetic control ofcoleopteran pest infestations in host organisms. More particularly, thepresent invention includes the methods for delivery of pest controlagents to a coleopteran pest. Such pest control agents cause, directlyor indirectly, an impairment in the ability of the pest to maintainitself, grow or otherwise infest a target host or symbiont. The presentinvention provides methods for employing stabilized dsRNA molecules inthe diet of the pest as a means for suppression of targeted genes in thepest, thus achieving desired control of pest infestations in, or aboutthe host or symbiont targeted by the pest.

In accomplishing the foregoing, the present invention provides a methodof inhibiting expression of a target gene in a coleopteran pest,including for example, corn rootworms or other coleopteran insectspecies, resulting in the cessation of feeding, growth, development,reproduction, infectivity, and eventually may result in the death of thepest. The method comprises in one embodiment introducing partial orfully stabilized double-stranded RNA (dsRNA) nucleotide molecules into anutritional composition that the pest relies on as a food source, andmaking the nutritional composition available to the pest for feeding.Ingestion of the nutritional composition containing the double strandedor siRNA molecules results in the uptake of the molecules by the cellsof the pest, resulting in the inhibition of expression of at least onetarget gene in the cells of the pest. Inhibition of the target geneexerts a deleterious effect upon the pest.

In certain embodiments, dsRNA molecules provided by the inventioncomprise nucleotide sequences complementary to a sequence as set forthin any of SEQ ID NO:1 through SEQ ID NO:906, the inhibition of which ina pest organism results in the reduction or removal of a protein ornucleotide sequence agent that is essential for the pests' growth anddevelopment or other biological function. The nucleotide sequenceselected may exhibit from about 80% to at least about 100% sequenceidentity to one of the nucleotide sequences as set forth in SEQ ID NO:1through SEQ ID NO:906, as set forth in the sequence listing, includingthe complement thereof. Such inhibition can be described as specific inthat a nucleotide sequence from a portion of the target gene is chosenfrom which the inhibitory dsRNA or siRNA is transcribed. The method iseffective in inhibiting the expression of at least one target gene andcan be used to inhibit many different types of target genes in the pest.In particular embodiments, the nucleotide sequence may be selected fromthe group consisting of SEQ ID NO:697, SEQ ID NOs:813-819, SEQ IDNO:841, and SEQ ID NO:874.

The sequences identified as having a pest protective effect may bereadily expressed as dsRNA molecules through the creation of appropriateexpression constructs. For example, such sequences can be expressed as ahairpin and stem and loop structure by taking a first segmentcorresponding to a sequence selected from SEQ ID NO:1 through SEQ IDNO:906 or a fragment thereof, linking this sequence to a second segmentspacer region that is not homologous or complementary to the firstsegment, and linking this to a third segment that transcribes an RNA,wherein at least a portion of the third segment is substantiallycomplementary to the first segment. Such a construct forms a stem andloop structure by hybridization of the first segment with the thirdsegment and a loop structure forms comprising the second segment(WO94/01550, WO98/05770, US 2002/0048814A1, and US 2003/0018993A1).

A. Nucleic Acid Compositions and Constructs

The invention provides recombinant DNA constructs for use in achievingstable transformation of particular host or symbiont pest targets.Transformed host or symbiont pest targets may express pesticidallyeffective levels of preferred dsRNA or siRNA molecules from therecombinant DNA constructs, and provide the molecules in the diet of thepest. Pairs of isolated and purified nucleotide sequences may beprovided from cDNA library and/or genomic library information. The pairsof nucleotide sequences may be derived from any preferred coleopteranpest for use as thermal amplification primers to generate DNA templatesfor the preparation of dsRNA and siRNA molecules of the presentinvention.

As used herein, the term “nucleic acid” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. The “nucleic acid” may also optionallycontain non-naturally occurring or altered nucleotide bases that permitcorrect read through by a polymerase and do not reduce expression of apolypeptide encoded by that nucleic acid. The term “nucleotide sequence”or “nucleic acid sequence” refers to both the sense and antisensestrands of a nucleic acid as either individual single strands or in theduplex. The term “ribonucleic acid” (RNA) is inclusive of RNAi(inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interferingRNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA,whether charged or discharged with a corresponding acylated amino acid),and cRNA (complementary RNA) and the term “deoxyribonucleic acid” (DNA)is inclusive of cDNA and genomic DNA and DNA-RNA hybrids. The words“nucleic acid segment”, “nucleotide sequence segment”, or more generally“segment” will be understood by those in the art as a functional termthat includes both genomic sequences, ribosomal RNA sequences, transferRNA sequences, messenger RNA sequences, operon sequences and smallerengineered nucleotide sequences that express or may be adapted toexpress, proteins, polypeptides or peptides.

Provided according to the invention are nucleotide sequences, theexpression of which results in an RNA sequence which is substantiallyhomologous to an RNA molecule of a targeted gene in an insect thatcomprises an RNA sequence encoded by a nucleotide sequence within thegenome of the insect. Thus, after ingestion of the stabilized RNAsequence down-regulation of the nucleotide sequence of the target genein the cells of the insect may be obtained resulting in a deleteriouseffect on the maintenance, viability, proliferation, reproduction andinfestation of the insect.

As used herein, the term “substantially homologous” or “substantialhomology”, with reference to a nucleic acid sequence, includes anucleotide sequence that hybridizes under stringent conditions to thecoding sequence as set forth in any of SEQ ID NO:1 through SEQ ID NO:906as set forth in the sequence listing, or the complements thereof.Sequences that hybridize under stringent conditions to any of SEQ IDNO:1 through SEQ ID NO:906 as set forth in the sequence listing, or thecomplements thereof, are those that allow an antiparallel alignment totake place between the two sequences, and the two sequences are thenable, under stringent conditions, to form hydrogen bonds withcorresponding bases on the opposite strand to form a duplex moleculethat is sufficiently stable under the stringent conditions to bedetectable using methods well known in the art. Substantially homologoussequences have preferably from about 70% to about 80% sequence identity,or more preferably from about 80% to about 85% sequence identity, ormost preferable from about 90% to about 95% sequence identity, to about99% sequence identity, to the referent nucleotide sequences as set forthin any of SEQ ID NO:1 through SEQ ID NO:906 as set forth in the sequencelisting, or the complements thereof.

As used herein, the term “sequence identity”, “sequence similarity” or“homology” is used to describe sequence relationships between two ormore nucleotide sequences. The percentage of “sequence identity” betweentwo sequences is determined by comparing two optimally aligned sequencesover a comparison window, wherein the portion of the sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison,and multiplying the result by 100 to yield the percentage of sequenceidentity. A sequence that is identical at every position in comparisonto a reference sequence is said to be identical to the referencesequence and vice-versa. A first nucleotide sequence when observed inthe 5′ to 3′ direction is said to be a “complement” of, or complementaryto, a second or reference nucleotide sequence observed in the 3′ to 5′direction if the first nucleotide sequence exhibits completecomplementarity with the second or reference sequence. As used herein,nucleic acid sequence molecules are said to exhibit “completecomplementarity” when every nucleotide of one of the sequences read 5′to 3′ is complementary to every nucleotide of the other sequence whenread 3′ to 5′. A nucleotide sequence that is complementary to areference nucleotide sequence will exhibit a sequence identical to thereverse complement sequence of the reference nucleotide sequence. Theseterms and descriptions are well defined in the art and are easilyunderstood by those of ordinary skill in the art.

As used herein, a “comparison window” refers to a conceptual segment ofat least 6 contiguous positions, usually about 50 to about 100, moreusually about 100 to about 150, in which a sequence is compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. The comparison window may compriseadditions or deletions (i.e. gaps) of about 20% or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences Those skilled in the artshould refer to the detailed methods used for sequence alignment in theWisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Drive Madison, Wis., USA) or refer to Ausubel et al.(1998) for a detailed discussion of sequence analysis.

The present invention provides DNA sequences capable of being expressedas an RNA in a cell or microorganism to inhibit target gene expressionin a cell, tissue or organ of an insect. The sequences comprises a DNAmolecule coding for one or more different nucleotide sequences, whereineach of the different nucleotide sequences comprises a sense nucleotidesequence and an antisense nucleotide sequence connected by a spacersequence coding for a dsRNA molecule of the present invention. Thespacer sequence constitutes part of the sense nucleotide sequence or theantisense nucleotide sequence and forms within the dsRNA moleculebetween the sense and antisense sequences. The sense nucleotide sequenceor the antisense nucleotide sequence is substantially identical to thenucleotide sequence of the target gene or a derivative thereof or acomplementary sequence thereto. The dsDNA molecule may be placedoperably under the control of a promoter sequence that functions in thecell, tissue or organ of the host expressing the dsDNA to produce dsRNAmolecules. In one embodiment, the DNA sequence may be derived from anucleotide sequence as set forth in SEQ ID NO:1 through SEQ ID NO:906 inthe sequence listing.

The invention also provides a DNA sequence for expression in a cell of aplant that, upon expression of the DNA to RNA and ingestion by a targetpest achieves suppression of a target gene in a cell, tissue or organ ofan insect pest. The dsRNA at least comprises one or multiple structuralgene sequences, wherein each of the structural gene sequences comprisesa sense nucleotide sequence and an antisense nucleotide sequenceconnected by a spacer sequence that forms a loop within thecomplementary and antisense sequences. The sense nucleotide sequence orthe antisense nucleotide sequence is substantially identical to thenucleotide sequence of the target gene, derivative thereof, or sequencecomplementary thereto. The one or more structural gene sequences isplaced operably under the control of one or more promoter sequences, atleast one of which is operable in the cell, tissue or organ of aprokaryotic or eukaryotic organism, particularly a plant.

A gene sequence or fragment for pest control according to the inventionmay be cloned between two tissue specific promoters, such as two rootspecific promoters which are operable in a transgenic plant cell andtherein expressed to produce mRNA in the transgenic plant cell that formdsRNA molecules thereto. The dsRNA molecules contained in plant tissuesare ingested by an insect so that the intended suppression of the targetgene expression is achieved.

A nucleotide sequence provided by the present invention may comprise aninverted repeat separated by a “spacer sequence.” The spacer sequencemay be a region comprising any sequence of nucleotides that facilitatessecondary structure formation between each repeat, where this isrequired. In one embodiment of the present invention, the spacersequence is part of the sense or antisense coding sequence for mRNA. Thespacer sequence may alternatively comprise any combination ofnucleotides or homologues thereof that are capable of being linkedcovalently to a nucleic acid molecule. The spacer sequence may comprisea sequence of nucleotides of at least about 10-100 nucleotides inlength, or alternatively at least about 100-200 nucleotides in length,at least 200-400 about nucleotides in length, or at least about 400-500nucleotides in length.

The nucleic acid molecules or fragment of the nucleic acid molecules orother nucleic acid molecules in the sequence listing are capable ofspecifically hybridizing to other nucleic acid molecules under certaincircumstances. As used herein, two nucleic acid molecules are said to becapable of specifically hybridizing to one another if the two moleculesare capable of forming an anti-parallel, double-stranded nucleic acidstructure. A nucleic acid molecule is said to be the complement ofanother nucleic acid molecule if they exhibit complete complementarity.Two molecules are said to be “minimally complementary” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to becomplementary if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook, et al. (1989), and by Haymes etal. (1985).

Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. Thus, in order for anucleic acid molecule or a fragment of the nucleic acid molecule toserve as a primer or probe it needs only be sufficiently complementaryin sequence to be able to form a stable double-stranded structure underthe particular solvent and salt concentrations employed.

Appropriate stringency conditions which promote DNA hybridization are,for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology(1989). For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. A nucleic acid for use in the present invention mayspecifically hybridize to one or more of nucleic acid molecules from WCRor complements thereof under such conditions. Preferably, a nucleic acidfor use in the present invention will exhibit at least from about 80%,or at least from about 90%, or at least from about 95%, or at least fromabout 98% or even about 100% sequence identity with one or more nucleicacid molecules as set forth in SEQ ID NO:1 through SEQ ID NO:906 as setforth in the sequence listing.

Nucleic acids of the present invention may also be synthesized, eithercompletely or in part, especially where it is desirable to provideplant-preferred sequences, by methods known in the art. Thus, all or aportion of the nucleic acids of the present invention may be synthesizedusing codons preferred by a selected host. Species-preferred codons maybe determined, for example, from the codons used most frequently in theproteins expressed in a particular host species. Other modifications ofthe nucleotide sequences may result in mutants having slightly alteredactivity.

dsRNA or siRNA nucleotide sequences comprise double strands ofpolymerized ribonucleotide and may include modifications to either thephosphate-sugar backbone or the nucleoside. Modifications in RNAstructure may be tailored to allow specific genetic inhibition. In oneembodiment, the dsRNA molecules may be modified through an enzymaticprocess so that siRNA molecules may be generated. The siRNA canefficiently mediate the down-regulation effect for some target genes insome insects. This enzymatic process may be accomplished by utilizing anRNAse III enzyme or a DICER enzyme, present in the cells of an insect, avertebrate animal, a fungus or a plant in the eukaryotic RNAi pathway(Elbashir et al., 2002; Hamilton and Baulcombe, 1999). This process mayalso utilize a recombinant DICER or RNAse III introduced into the cellsof a target insect through recombinant DNA techniques that are readilyknown to the skilled in the art. Both the DICER enzyme and RNAse III,being naturally occurring in an insect or being made through recombinantDNA techniques, cleave larger dsRNA strands into smalleroligonucleotides. The DICER enzymes specifically cut the dsRNA moleculesinto siRNA pieces each of which is about 19-25 nucleotides in lengthwhile the RNAse III enzymes normally cleave the dsRNA molecules into12-15 base-pair siRNA. The siRNA molecules produced by the either of theenzymes have 2 to 3 nucleotide 3′ overhangs, and 5′ phosphate and 3′hydroxyl termini. The siRNA molecules generated by RNAse III enzyme arethe same as those produced by Dicer enzymes in the eukaryotic RNAipathway and are hence then targeted and degraded by an inherent cellularRNA-degrading mechanism after they are subsequently unwound, separatedinto single-stranded RNA and hybridize with the RNA sequencestranscribed by the target gene. This process results in the effectivedegradation or removal of the RNA sequence encoded by the nucleotidesequence of the target gene in the insect. The outcome is the silencingof a particularly targeted nucleotide sequence within the insect.Detailed descriptions of enzymatic processes can be found in Hannon(2002).

A nucleotide sequence of the present invention can be recorded oncomputer readable media. As used herein, “computer readable media”refers to any tangible medium of expression that can be read andaccessed directly by a computer. Such media include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc, storagemedium, and magnetic tape: optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; optical characterrecognition formatted computer files, and hybrids of these categoriessuch as magnetic/optical storage media. A skilled artisan can readilyappreciate that any of the presently known computer readable mediums canbe used to create a manufacture comprising computer readable mediumhaving recorded thereon a nucleotide sequence of the present invention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable medium to generate media comprising the nucleotide sequenceinformation of the present invention. A variety of data storagestructures are available to a skilled artisan for creating a computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable medium. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and Microsoft Word,or represented in the form of an ASCII text file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. The skilledartisan can readily adapt any number of data processor structuringformats (e.g. text file or database) in order to obtain computerreadable medium having recorded thereon the nucleotide sequenceinformation of the present invention.

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable medium.Software that implements the BLAST (Altschul et al., 1990) and BLAZE(Brutlag, et al., 1993) search algorithms on a Sybase system can be usedto identify open reading frames (ORFs) within sequences such as theUnigenes and EST's that are provided herein and that contain homology toORFs or proteins from other organisms. Such ORFs are protein-encodingfragments within the sequences of the present invention and are usefulin producing commercially important proteins such as enzymes used inamino acid biosynthesis, metabolism, transcription, translation, RNAprocessing, nucleic acid and a protein degradation, proteinmodification, and DNA replication, restriction, modification,recombination, and repair.

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence information describedherein. Such systems are designed to identify commercially importantfragments of the nucleic acid molecule of the present invention. As usedherein, “a computer-based system” refers to the hardware means, softwaremeans, and data storage means used to analyze the nucleotide sequenceinformation of the present invention. The minimum hardware means of thecomputer-based systems of the present invention comprises a centralprocessing unit (CPU), input means, output means, and data storagemeans. A skilled artisan can readily appreciate that any one of thecurrently available computer-based system are suitable for use in thepresent invention.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequences or sequence(s) are chosen based on a three-dimensionalconfiguration that is formed upon the folding of the target motif. Thereare a variety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzymatic active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, cis elements, hairpin structures and inducibleexpression elements (protein binding sequences).

B. Recombinant Vectors and Host Cell Transformation

A recombinant DNA vector may, for example, be a linear or a closedcircular plasmid. The vector system may be a single vector or plasmid ortwo or more vectors or plasmids that together contain the total DNA tobe introduced into the genome of the bacterial host. In addition, abacterial vector may be an expression vector. Nucleic acid molecules asset forth in SEQ ID NO:1 through SEQ ID NO:906 or fragments orcomplements thereof can, for example, be suitably inserted into a vectorunder the control of a suitable promoter that functions in one or moremicrobial hosts to drive expression of a linked coding sequence or otherDNA sequence. Many vectors are available for this purpose, and selectionof the appropriate vector will depend mainly on the size of the nucleicacid to be inserted into the vector and the particular host cell to betransformed with the vector. Each vector contains various componentsdepending on its function (amplification of DNA or expression of DNA)and the particular host cell with which it is compatible. The vectorcomponents for bacterial transformation generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more selectable marker genes, and an induciblepromoter allowing the expression of exogenous DNA.

Expression and cloning vectors generally contain a selection gene, alsoreferred to as a selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.Those cells that are successfully transformed with a heterologousprotein or fragment thereof produce a protein conferring drug resistanceand thus survive the selection regimen.

An expression vector for producing a mRNA can also contain an induciblepromoter that is recognized by the host bacterial organism and isoperably linked to the nucleic acid encoding, for example, the nucleicacid molecule coding the D. v. virgifera mRNA or fragment thereof ofinterest. Inducible promoters suitable for use with bacterial hostsinclude β-lactamase promoter, E. coli λ phage PL and PR promoters, andE. coli galactose promoter, arabinose promoter, alkaline phosphatasepromoter, tryptophan (trp) promoter, and the lactose operon promoter andvariations thereof and hybrid promoters such as the tac promoter.However, other known bacterial inducible promoters are suitable.

The term “operably linked”, as used in reference to a regulatorysequence and a structural nucleotide sequence, means that the regulatorysequence causes regulated expression of the linked structural nucleotidesequence. “Regulatory sequences” or “control elements” refer tonucleotide sequences located upstream (5′ noncoding sequences), within,or downstream (3′ non-translated sequences) of a structural nucleotidesequence, and which influence the timing and level or amount oftranscription, RNA processing or stability, or translation of theassociated structural nucleotide sequence. Regulatory sequences mayinclude promoters, translation leader sequences, introns, enhancers,stem-loop structures, repressor binding sequences, and polyadenylationrecognition sequences and the like.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectorstypically contain at least one sequence homologous to the bacterialchromosome that allows the vector to integrate. Integrations appear toresult from recombinations between homologous DNA in the vector and thebacterial chromosome. For example, integrating vectors constructed withDNA from various Bacillus strains integrate into the Bacillus chromosome(EP 0 127,328). Integrating vectors may also be comprised ofbacteriophage or transposon sequences. Suicide vectors are also known inthe art.

Construction of suitable vectors containing one or more of theabove-listed components employs standard recombinant DNA techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to generate the plasmids required. Examples ofavailable bacterial expression vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asBluescript™ (Stratagene, La Jolla, Calif.), in which, for example, a D.v. virgifera protein or fragment thereof, may be ligated into the vectorin frame with sequences for the amino-terminal Met and the subsequent 7residues of β-galactosidase so that a hybrid protein is produced; pINvectors (Van Heeke and Schuster, 1989); and the like.

A yeast recombinant construct can typically include one or more of thefollowing: a promoter sequence, fusion partner sequence, leadersequence, transcription termination sequence, a selectable marker. Theseelements can be combined into an expression cassette, which may bemaintained in a replicon, such as an extrachromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as yeast orbacteria. The replicon may have two replication systems, thus allowingit to be maintained, for example, in yeast for expression and in aprokaryotic host for cloning and amplification. Examples of suchyeast-bacteria shuttle vectors include YEp24 (Botstein et al., 1979),pC1/1 (Brake et al., 1984), and YRp17 (Stinchcomb et al., 1982). Inaddition, a replicon may be either a high or low copy number plasmid. Ahigh copy number plasmid will generally have a copy number ranging fromabout 5 to about 200, and typically about 10 to about 150. A hostcontaining a high copy number plasmid will preferably have at leastabout 10, and more preferably at least about 20.

Useful yeast promoter sequences can be derived from genes encodingenzymes in the metabolic pathway. Examples of such genes include alcoholdehydrogenase (ADH) (EP 0 284044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK) (EP 0 3215447). The yeast PHO5 gene,encoding acid phosphatase, also provides useful promoter sequences(Myanohara et al., 1983). In addition, synthetic promoters that do notoccur in nature also function as yeast promoters. Examples of suchhybrid promoters include the ADH regulatory sequence linked to the GAPtranscription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Examples of transcription terminator sequences and otheryeast-recognized termination sequences, such as those coding forglycolytic enzymes, are known to those of skill in the art.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors typicallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome (Orr-Weaver et al., 1983). An integrating vector may bedirected to a specific locus in yeast by selecting the appropriatehomologous sequence for inclusion in the vector. See Orr-Weaver et al.,supra. One or more expression constructs may integrate, possiblyaffecting levels of recombinant protein produced (Rine et al., 1983).

The present invention also contemplates transformation of a nucleotidesequence of the present invention into a plant to achieve pestinhibitory levels of expression of one or more dsRNA molecules. Atransformation vector can be readily prepared using methods available inthe art. The transformation vector comprises one or more nucleotidesequences that is/are capable of being transcribed to an RNA moleculeand that is/are substantially homologous and/or complementary to one ormore nucleotide sequences encoded by the genome of the insect, such thatupon uptake of the RNA there is down-regulation of expression of atleast one of the respective nucleotide sequences of the genome of theinsect.

The transformation vector may be termed a dsDNA construct and may alsobe defined as a recombinant molecule, an insect control agent, a geneticmolecule or a chimeric genetic construct. A chimeric genetic constructof the present invention may comprise, for example, nucleotide sequencesencoding one or more antisense transcripts, one or more sensetranscripts, one or more of each of the aforementioned, wherein all orpart of a transcript therefrom is homologous to all or part of an RNAmolecule comprising an RNA sequence encoded by a nucleotide sequencewithin the genome of an insect.

In one embodiment the plant transformation vector comprises an isolatedand purified DNA molecule comprising a promoter operatively linked toone or more nucleotide sequences of the present invention. Thenucleotide sequence is selected from the group consisting of SEQ ID NO:1through SEQ ID NO:906 as set forth in the sequence listing. Thenucleotide sequence includes a segment coding all or part of an RNApresent within a targeted pest RNA transcript and may comprise invertedrepeats of all or a part of a targeted pest RNA. The DNA moleculecomprising the expression vector may also contain a functional intronsequence positioned either upstream of the coding sequence or evenwithin the coding sequence, and may also contain a five prime (5′)untranslated leader sequence (i.e., a UTR or 5′-UTR) positioned betweenthe promoter and the point of translation initiation.

A plant transformation vector may contain sequences from more than onegene, thus allowing production of more than one dsRNA for inhibitingexpression of two or more genes in cells of a target pest. One skilledin the art will readily appreciate that segments of DNA whose sequencecorresponds to that present in different genes can be combined into asingle composite DNA segment for expression in a transgenic plant.Alternatively, a plasmid of the present invention already containing atleast one DNA segment can be modified by the sequential insertion ofadditional DNA segments between the enhancer and promoter and terminatorsequences. In the insect control agent of the present invention designedfor the inhibition of multiple genes, the genes to be inhibited can beobtained from the same insect species in order to enhance theeffectiveness of the insect control agent. In certain embodiments, thegenes can be derived from different insects in order to broaden therange of insects against which the agent is effective. When multiplegenes are targeted for suppression or a combination of expression andsuppression, a polycistronic DNA element can be fabricated asillustrated and disclosed in Fillatti, Application Publication No. US2004-0029283.

Promoters that function in different plant species are also well knownin the art. Promoters useful for expression of polypeptides in plantsinclude those that are inducible, viral, synthetic, or constitutive asdescribed in Odell et al. (1985), and/or promoters that are temporallyregulated, spatially regulated, and spatio-temporally regulated.Preferred promoters include the enhanced CaMV35S promoters, and theFMV35S promoter. For the purpose of the present invention, e.g., foroptimum control of species that feed on roots, it may be preferable toachieve the highest levels of expression of these genes within the rootsof plants. A number of root-enhanced promoters have been identified andare known in the art (Lu et al., 2000; U.S. Pat. Nos. 5,837,848 and6,489,542).

A recombinant DNA vector or construct of the present invention willtypically comprise a selectable marker that confers a selectablephenotype on plant cells. Selectable markers may also be used to selectfor plants or plant cells that contain the exogenous nucleic acidsencoding polypeptides or proteins of the present invention. The markermay encode biocide resistance, antibiotic resistance (e.g., kanamycin,G418 bleomycin, hygromycin, etc.), or herbicide resistance (e.g.,glyphosate, etc.). Examples of selectable markers include, but are notlimited to, a neo gene which codes for kanamycin resistance and can beselected for using kanamycin, G418, etc., a bar gene which codes forbialaphos resistance; a mutant EPSP synthase gene which encodesglyphosate resistance; a nitrilase gene which confers resistance tobromoxynil; a mutant acetolactate synthase gene (ALS) which confersimidazolinone or sulfonylurea resistance; and a methotrexate resistantDHFR gene. Examples of such selectable markers are illustrated in U.S.Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047.

A recombinant vector or construct of the present invention may alsoinclude a screenable marker. Screenable markers may be used to monitorexpression. Exemplary screenable markers include a β-glucuronidase oruidA gene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known (Jefferson, 1987; Jefferson et al., 1987); anR-locus gene, which encodes a product that regulates the production ofanthocyanin pigments (red color) in plant tissues (Dellaporta et al.,1988); a β-lactamase gene (Sutcliffe et al., 1978), a gene which encodesan enzyme for which various chromogenic substrates are known (e.g.,PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al., 1986)a xylE gene (Zukowsky et al., 1983) which encodes a catechol dioxygenasethat can convert chromogenic catechols; an α-amylase gene (Ikatu et al.,1990); a tyrosinase gene (Katz et al., 1983) which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to melanin; an α-galactosidase, which catalyzes a chromogenicα-galactose substrate.

Preferred plant transformation vectors include those derived from a Tiplasmid of Agrobacterium tumefaciens (e.g. U.S. Pat. Nos. 4,536,475,4,693,977, 4,886,937, 5,501,967 and EP 0 122 791). Agrobacteriumrhizogenes plasmids (or “Ri”) are also useful and known in the art.Other preferred plant transformation vectors include those disclosed,e.g., by Herrera-Estrella (1983); Bevan (1983), Klee (1985) and EP 0 120516.

In general it is preferred to introduce a functional recombinant DNA ata non-specific location in a plant genome. In special cases it may beuseful to insert a recombinant DNA construct by site-specificintegration. Several site-specific recombination systems exist which areknown to function implants include cre-lox as disclosed in U.S. Pat. No.4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695.

Suitable methods for transformation of host cells for use with thecurrent invention are believed to include virtually any method by whichDNA can be introduced into a cell, such as by direct delivery of DNAsuch as by PEG-mediated transformation of protoplasts (Omirulleh et al.,1993), by desiccation/inhibition-mediated DNA uptake (Potrykus et al.,1985), by electroporation (U.S. Pat. No. 5,384,253), by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523;and U.S. Pat. No. 5,464,765), by Agrobacterium-mediated transformation(U.S. Pat. No. 5,591,616 and U.S. Pat. No. 5,563,055) and byacceleration of DNA coated particles (U.S. Pat. No. 5,550,318; U.S. Pat.No. 5,538,877; and U.S. Pat. No. 5,538,880), etc. Through theapplication of techniques such as these, the cells of virtually anyspecies may be stably transformed. In the case of multicellular species,the transgenic cells may be regenerated into transgenic organisms.

Methods for the creation of transgenic plants and expression ofheterologous nucleic acids in plants in particular are known and may beused with the nucleic acids provided herein to prepare transgenic plantsthat exhibit reduced susceptibility to feeding by a target pest organismsuch as corn rootworms. Plant transformation vectors can be prepared,for example, by inserting the dsRNA producing nucleic acids disclosedherein into plant transformation vectors and introducing these intoplants. One known vector system has been derived by modifying thenatural gene transfer system of Agrobacterium tumefaciens. The naturalsystem comprises large Ti (tumor-inducing)-plasmids containing a largesegment, known as T-DNA, which is transferred to transformed plants.Another segment of the Ti plasmid, the vir region, is responsible forT-DNA transfer. The T-DNA region is bordered by terminal repeats. In themodified binary vectors the tumor-inducing genes have been deleted andthe functions of the vir region are utilized to transfer foreign DNAbordered by the T-DNA border sequences. The T-region may also contain aselectable marker for efficient recovery of transgenic plants and cells,and a multiple cloning site for inserting sequences for transfer such asa dsRNA encoding nucleic acid.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single simple recombinant DNA sequence insertedinto one chromosome and is referred to as a transgenic event. Suchtransgenic plants can be referred to as being heterozygous for theinserted exogenous sequence. A transgenic plant homozygous with respectto a transgene can be obtained by selfing an independent segreganttransgenic plant to produce F1 seed. One fourth of the F1 seed producedwill be homozygous with respect to the transgene. Germinating F1 seedresults in plants that can be tested for heterozygosity or homozygosity,typically using a SNP assay or a thermal amplification assay that allowsfor the distinction between heterozygotes and homozygotes (i.e., azygosity assay).

C. Nucleic Acid Expression and Target Gene Suppression

The present invention provides, as an example, a transformed host orsymbiont pest target organism, transformed plant cells and transformedplants and their progeny. The transformed plant cells and transformedplants may be engineered to express one or more of the dsRNA or siRNAsequences described herein to provide a pest-protective effect. Thesesequences may be used for gene suppression in a pest organism, therebyreducing the predation by the pest on a protected transformed host orsymbiont organism. As used herein the words “gene suppression” areintended to refer to any of the well-known methods for reducing thelevels of gene transcription to mRNA and/or subsequent translation ofthe mRNA.

Gene suppression is also intended to mean the reduction of proteinexpression from a gene or a coding sequence includingposttranscriptional gene suppression and transcriptional suppression.Posttranscriptional gene suppression is mediated by the homology betweenof all or a part of a mRNA transcribed from a gene or coding sequencetargeted for suppression and the corresponding double stranded RNA usedfor suppression, and refers to the substantial and measurable reductionof the amount of available mRNA available in the cell for binding byribosomes. The transcribed RNA can be in the sense orientation to effectwhat is called co-suppression, in the anti-sense orientation to effectwhat is called anti-sense suppression, or in both orientations producinga dsRNA to effect what is called RNA interference (RNAi).

Transcriptional suppression is mediated by the presence in the cell of adsRNA gene suppression agent exhibiting substantial sequence identity toa promoter DNA sequence or the complement thereof to effect what isreferred to as promoter trans suppression. Gene suppression may beeffective against a native plant gene associated with a trait, e.g., toprovide plants with reduced levels of a protein encoded by the nativegene or with enhanced or reduced levels of an affected metabolite. Genesuppression can also be effective against target genes in plant peststhat may ingest or contact plant material containing gene suppressionagents, specifically designed to inhibit or suppress the expression ofone or more homologous or complementary sequences in the cells of thepest. Post-transcriptional gene suppression by anti-sense or senseoriented RNA to regulate gene expression in plant cells is disclosed inU.S. Pat. Nos. 5,107,065, 5,759,829, 5,283,184, and 5,231,020. The useof dsRNA to suppress genes in plants is disclosed in WO 99/53050, WO99/49029, U.S. Patent Application Publication No. 2003/0175965, and2003/0061626, U.S. patent application Ser. No. 10/465,800, and U.S. Pat.Nos. 6,506,559, and 6,326,193.

A beneficial method of post transcriptional gene suppression in plantsemploys both sense-oriented and anti-sense-oriented, transcribed RNAwhich is stabilized, e.g., as a hairpin and stem and loop structure. Apreferred DNA construct for effecting post transcriptional genesuppression is one in which a first segment encodes an RNA exhibiting ananti-sense orientation exhibiting substantial identity to a segment of agene targeted for suppression, which is linked to a second segment insense orientation encoding an RNA exhibiting substantial complementarityto the first segment. Such a construct forms a stem and loop structureby hybridization of the first segment with the second segment and a loopstructure from the nucleotide sequences linking the two segments (seeWO94/01550, WO98/05770, US 2002/0048814, and US 2003/0018993).

According to one embodiment of the present invention, there is provideda nucleotide sequence, for which in vitro expression results intranscription of a stabilized RNA sequence that is substantiallyhomologous to an RNA molecule of a targeted gene in an insect thatcomprises an RNA sequence encoded by a nucleotide sequence within thegenome of the insect. Thus, after the insect ingests the stabilized RNAsequence incorporated in a diet or sprayed on a plant surface, adown-regulation of the nucleotide sequence corresponding to the targetgene in the cells of a target insect is affected.

Inhibition of a target gene using the stabilized dsRNA technology of thepresent invention is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. RNA containing a nucleotide sequences identical to a portionof the target gene is preferred for inhibition. RNA sequences withinsertions, deletions, and single point mutations relative to the targetsequence have also been found to be effective for inhibition. Inperformance of the present invention, it is preferred that theinhibitory dsRNA and the portion of the target gene share at least fromabout 80% sequence identity, or from about 90% sequence identity, orfrom about 95% sequence identity, or from about 99% sequence identity,or even about 100% sequence identity. Alternatively, the duplex regionof the RNA may be defined functionally as a nucleotide sequence that iscapable of hybridizing with a portion of the target gene transcript. Aless than full length sequence exhibiting a greater homology compensatesfor a longer less homologous sequence. The length of the identicalnucleotide sequences may be at least about 25, 50, 100, 200, 300, 400,500 or at least about 1000 bases. Normally, a sequence of greater than20-100 nucleotides should be used, though a sequence of greater thanabout 200-300 nucleotides would be preferred, and a sequence of greaterthan about 500-1000 nucleotides would be especially preferred dependingon the size of the target gene. The invention has the advantage of beingable to tolerate sequence variations that might be expected due togenetic mutation, strain polymorphism, or evolutionary divergence. Theintroduced nucleic acid molecule may not need to be absolute homology,may not need to be full length, relative to either the primarytranscription product or fully processed mRNA of the target gene.Therefore, those skilled in the art need to realize that, as disclosedherein, 100% sequence identity between the RNA and the target gene isnot required to practice the present invention.

Inhibition of target gene expression may be quantified by measuringeither the endogenous target RNA or the protein produced by translationof the target RNA and the consequences of inhibition can be confirmed byexamination of the outward properties of the cell or organism.Techniques for quantifying RNA and proteins are well known to one ofordinary skill in the art. Multiple selectable markers are availablethat confer resistance to ampicillin, bleomycin, chloramphenicol,gentamycin, hygromycin, kanamycin, lincomycin, methotrexate,phosphinothricin, puromycin, spectinomycin, rifampicin, and tetracyclin,and the like.

In certain embodiments gene expression is inhibited by at least 10%,preferably by at least 33%, more preferably by at least 50%, and yetmore preferably by at least 80%. In particularly preferred embodimentsof the invention gene expression is inhibited by at least 80%, morepreferably by at least 90%, more preferably by at least 95%, or by atleast 99% within cells in the insect so a significant inhibition takesplace. Significant inhibition is intended to refer to sufficientinhibition that results in a detectable phenotype (e.g., cessation oflarval growth, paralysis or mortality, etc.) or a detectable decrease inRNA and/or protein corresponding to the target gene being inhibited.Although in certain embodiments of the invention inhibition occurs insubstantially all cells of the insect, in other preferred embodimentsinhibition occurs in only a subset of cells expressing the gene. Forexample, if the gene to be inhibited plays an essential role in cells inthe insect alimentary tract, inhibition of the gene within these cellsis sufficient to exert a deleterious effect on the insect.

dsRNA molecules may be synthesized either in vivo or in vitro. The dsRNAmay be formed by a single self-complementary RNA strand or from twocomplementary RNA strands. Endogenous RNA polymerase of the cell maymediate transcription in vivo, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. Inhibition may be targeted byspecific transcription in an organ, tissue, or cell type; stimulation ofan environmental condition (e.g., infection, stress, temperature,chemical inducers); and/or engineering transcription at a developmentalstage or age. The RNA strands may or may not be polyadenylated; the RNAstrands may or may not be capable of being translated into a polypeptideby a cell's translational apparatus.

A RNA, dsRNA, siRNA, or miRNA of the present invention may be producedchemically or enzymatically by one skilled in the art through manual orautomated reactions or in vivo in another organism. RNA may also beproduced by partial or total organic synthesis; any modifiedribonucleotide can be introduced by in vitro enzymatic or organicsynthesis. The RNA may be synthesized by a cellular RNA polymerase or abacteriophage RNA polymerase (e.g., T3, T7, SP6). The use and productionof an expression construct are known in the art (see, for example, WO97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and5,804,693). If synthesized chemically or by in vitro enzymaticsynthesis, the RNA may be purified prior to introduction into the cell.For example, RNA can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the RNA may be used with no or aminimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

For transcription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, andpolyadenylation) may be used to transcribe the RNA strand (or strands).Therefore, in one embodiment, the nucleotide sequences for use inproducing RNA molecules may be operably linked to one or more promotersequences functional in a microorganism, a fungus or a plant host cell.Ideally, the nucleotide sequences are placed under the control of anendogenous promoter, normally resident in the host genome. Thenucleotide sequence of the present invention, under the control of anoperably linked promoter sequence, may further be flanked by additionalsequences that advantageously affect its transcription and/or thestability of a resulting transcript. Such sequences are generallylocated upstream of the operably linked promoter and/or downstream ofthe 3′ end of the expression construct and may occur both upstream ofthe promoter and downstream of the 3′ end of the expression construct,although such an upstream sequence only is also contemplated.

As used herein, the term “insect control agent”, or “gene suppressionagent” refers to a particular RNA molecule comprising a first RNAsegment and a second RNA segment, wherein the complementarity betweenthe first and the second RNA segments results in the ability of the twosegments to hybridize in vivo and in vitro to form a double strandedmolecule. It may generally be preferable to include a third RNA segmentlinking and stabilizing the first and second sequences such that theentire structure forms into a stem and loop structure, or even moretightly hybridizing structures may form into a stem-loop knottedstructure. Alternatively, a symmetrical hairpin could be formed withouta third segment in which there is no designed loop, but for stericreasons a hairpin would create its own loop when the stem is long enoughto stabilize itself. The first and the second RNA segments willgenerally lie within the length of the RNA molecule and be substantiallyinverted repeats of each other and linked together by the third RNAsegment. The first and the second segments correspond invariably and notrespectively to a sense and an antisense sequence with respect to thetarget RNA transcribed from the target gene in the target insect pestthat is suppressed by the ingestion of the dsRNA molecule. The insectcontrol agent can also be a substantially purified (or isolated) nucleicacid molecule and more specifically nucleic acid molecules or nucleicacid fragment molecules thereof from a genomic DNA (gDNA) or cDNAlibrary. Alternatively, the fragments may comprise smalleroligonucleotides having from about 15 to about 250 nucleotide residues,and more preferably, about 15 to about 30 nucleotide residues.

As used herein, the term “genome” as it applies to cells of an insect ora host encompasses not only chromosomal DNA found within the nucleus,but organelle DNA found within subcellular components of the cell. TheDNA's of the present invention introduced into plant cells can thereforebe either chromosomally integrated or organelle-localized. The term“genome” as it applies to bacteria encompasses both the chromosome andplasmids within a bacterial host cell. The DNA's of the presentinvention introduced into bacterial host cells can therefore be eitherchromosomally integrated or plasmid-localized.

As used herein, the term “pest” refers to insects, arachnids,crustaceans, fungi, bacteria, viruses, nematodes, flatworms, roundworms,pinworms, hookworms, tapeworms, trypanosomes, schistosomes, botflies,fleas, ticks, mites, and lice and the like that are pervasive in thehuman environment and that may ingest or contact one or more cells,tissues, or fluids produced by a pest host or symbiont transformed toexpress or coated with a double stranded gene suppression agent or thatmay ingest plant material containing the gene suppression agent. As usedherein, a “pest resistance” trait is a characteristic of a transgenicplant, transgenic animal, transgenic host or transgenic symbiont thatcauses the plant, animal, host, or symbiont to be resistant to attackfrom a pest that typically is capable of inflicting damage or loss tothe plant, animal, host or symbiont. Such pest resistance can arise froma natural mutation or more typically from incorporation of recombinantDNA that confers pest resistance. To impart insect resistance to atransgenic plant a recombinant DNA can, for example, be transcribed intoa RNA molecule that forms a dsRNA molecule within the tissues or fluidsof the recombinant plant. The dsRNA molecule is comprised in part of asegment of RNA that is identical to a corresponding RNA segment encodedfrom a DNA sequence within an insect pest that prefers to feed on therecombinant plant. Expression of the gene within the target insect pestis suppressed by the dsRNA, and the suppression of expression of thegene in the target insect pest results in the plant being insectresistant. Fire et al. (U.S. Pat. No. 6,506,599) generically describedinhibition of pest infestation, providing specifics only about severalnucleotide sequences that were effective for inhibition of gene functionin the nematode species Caenorhabditis elegans. Similarly, Plaetinck etal. (US 2003/0061626) describe the use of dsRNA for inhibiting genefunction in a variety of nematode pests. Mesa et al. (US 2003/0150017)describe using dsDNA sequences to transform host cells to expresscorresponding dsRNA sequences that are substantially identical to targetsequences in specific pathogens, and particularly describe constructingrecombinant plants expressing such dsRNA sequences for ingestion byvarious plant pests, facilitating down-regulation of a gene in thegenome of the pest and improving the resistance of the plant to the pestinfestation.

The present invention provides for inhibiting gene expression of one ormultiple target genes in a target pest using stabilized dsRNA methods.The invention is particularly useful in the modulation of eukaryoticgene expression, in particular the modulation of expression of genespresent in pests that exhibit a digestive system pH level that is fromabout 4.5 to about 9.5, more preferably from about 5.0 to about 8.0, andeven more preferably from about 6.5 to about 7.5. For plant pests with adigestive system that exhibits pH levels outside of these ranges,delivery methods may be desired for use that do not require ingestion ofdsRNA molecules.

The modulatory effect of dsRNA is applicable to a variety of genesexpressed in the pests including, for example, endogenous genesresponsible for cellular metabolism or cellular transformation,including house keeping genes, transcription factors and other geneswhich encode polypeptides involved in cellular metabolism.

As used herein, the phrase “inhibition of gene expression” or“inhibiting expression of a target gene in the cell of an insect” refersto the absence (or observable decrease) in the level of protein and/ormRNA product from the target gene. Specificity refers to the ability toinhibit the target gene without manifest effects on other genes of thecell and without any effects on any gene within the cell that isproducing the dsRNA molecule. The inhibition of gene expression of thetarget gene in the insect pest may result in novel phenotypic traits inthe insect pest.

The present invention provides in part a delivery system for thedelivery of the insect control agents to insects through their exposureto a diet containing the insect control agents of the present invention.In accordance with one of the embodiments, the stabilized dsRNA or siRNAmolecules may be incorporated in the insect diet or may be overlaid onthe top of the diet for consumption by an insect. The present inventionalso provides in part a delivery system for the delivery of the insectcontrol agents to insects through their exposure to a microorganism orhost such as a plant containing the insect control agents of the presentinvention by ingestion of the microorganism or the host cells or thecontents of the cells. In accordance with another embodiment, thepresent invention involves generating a transgenic plant cell or a plantthat contains a recombinant DNA construct transcribing the stabilizeddsRNA molecules of the present invention. As used herein, the phrase“generating a transgenic plant cell or a plant” refers to the methods ofemploying the recombinant DNA technologies readily available in the art(e.g., by Sambrook, et al., 1989) to construct a plant transformationvector transcribing the stabilized dsRNA molecules of the presentinvention, to transform the plant cell or the plant and to generate thetransgenic plant cell or the transgenic plant that contain thetranscribed, stabilized dsRNA molecules.

In still another embodiment, non-pathogenic, attenuated strains ofmicroorganisms may be used as a carrier for the insect control agentsand, in this perspective, the microorganisms carrying such agents arealso referred to as insect control agents. The microorganisms may beengineered to express a nucleotide sequence of a target gene to produceRNA molecules comprising RNA sequences homologous or complementary toRNA sequences typically found within the cells of an insect. Exposure ofthe insects to the microorganisms result in ingestion of themicroorganisms and down-regulation of expression of target genesmediated directly or indirectly by the RNA molecules or fragments orderivatives thereof.

The present invention alternatively provides exposure of an insect tothe insect control agents of the present invention incorporated in aspray mixer and applied to the surface of a host, such as a host plant.In an exemplary embodiment, ingestion of the insect control agents by aninsect delivers the insect control agents to the gut of the insect andsubsequently to the cells within the body of the insect. In anotherembodiment, infection of the insect by the insect control agents throughother means such as by injection or other physical methods also permitsdelivery of the insect control agents. In yet another embodiment, theRNA molecules themselves are encapsulated in a synthetic matrix such asa polymer and applied to the surface of a host such as a plant.Ingestion of the host cells by an insect permits delivery of the insectcontrol agents to the insect and results in down-regulation of a targetgene in the host.

It is envisioned that the compositions of the present invention can beincorporated within the seeds of a plant species either as a product ofexpression from a recombinant gene incorporated into a genome of theplant cells, or incorporated into a coating or seed treatment that isapplied to the seed before planting. The plant cell containing arecombinant gene is considered herein to be a transgenic event.

It is believed that a pesticidal seed treatment can provide significantadvantages when combined with a transgenic event that providesprotection from coleopteran pest infestation that is within thepreferred effectiveness range against a target pest. In addition, it isbelieved that there are situations that are well known to those havingskill in the art, where it is advantageous to have such transgenicevents within the preferred range of effectiveness.

The present invention provides in part a delivery system for thedelivery of insect control agents to insects. The stabilized dsRNA orsiRNA molecules of the present invention may be directly introduced intothe cells of an insect, or introduced into an extracellular cavity,interstitial space, lymph system, digestive system, into the circulationof the insect through oral ingestion or other means that one skilled inthe art may employ. Methods for oral introduction may include directmixing of RNA with food of the insect, as well as engineered approachesin which a species that is used as food is engineered to express thedsRNA or siRNA, then fed to the insect to be affected. In oneembodiment, for example, the dsRNA or siRNA molecules may beincorporated into, or overlaid on the top of, the insect's diet. Inanother embodiment, the RNA may be sprayed onto a plant surface. Instill another embodiment, the dsRNA or siRNA may be expressed bymicroorganisms and the microorganisms may be applied onto a plantsurface or introduced into a root, stem by a physical means such as aninjection. In still another embodiment, a plant may be geneticallyengineered to express the dsRNA or siRNA in an amount sufficient to killthe insects known to infect the plant.

Specifically, in practicing the present invention in WCR, the stabilizeddsRNA or siRNA may be introduced in the midgut inside the insect andachieve the desired inhibition of the targeted genes. The dsRNA or siRNAmolecules may be incorporated into a diet or be overlaid on the diet asdiscussed above and may be ingested by the insects. In any event, thedsRNA's of the present invention are provided in the diet of the targetpest. The target pest of the present invention will exhibit a digestivetract pH from about 4.5 to about 9.5, or from about 5 to about 8.5, orfrom about 6 to about 8, or from about 6.5 to about 7.7, or about 7.0.The digestive tract of a target pest is defined herein as the locationwithin the pest that food that is ingested by the target pest is exposedto an environment that is favorable for the uptake of the dsRNAmolecules of the present invention without suffering a pH so extremethat the hydrogen bonding between the double-strands of the dsRNA arecaused to dissociate and form single stranded molecules.

It is also anticipated that dsRNA's produced by chemical or enzymaticsynthesis may be formulated in a manner consistent with commonagricultural practices and used as spray-on products for controllinginsect infestations. The formulations may include the appropriatestickers and wetters required for efficient foliar coverage as well asUV protectants to protect dsRNAs from UV damage. Such additives arecommonly used in the bioinsecticide industry and are well known to thoseskilled in the art. Such applications could be combined with otherspray-on insecticide applications, biologically based or not, to enhanceplant protection from insect feeding damage.

The present inventors contemplate that bacterial strains producinginsecticidal proteins may be used to produce dsRNAs for insect controlpurposes. These strains may exhibit improved insect control properties.A variety of different bacterial hosts may be used to produce insectcontrol dsRNAs. Exemplary bacteria may include E. coli, B.thuringiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp.,Serratia entomophila and related Serratia sp., B. sphaericus, B. cereus,B. laterosporus, B. popilliae, Clostridium bifermentans and otherClostridium species, or other spore-forming gram-positive bacteria. Incertain embodiments, bacteria may be engineered for control of pestssuch as mosquitoes.

The present invention also relates to recombinant DNA constructs forexpression in a microorganism. Exogenous nucleic acids from which an RNAof interest is transcribed can be introduced into a microbial host cell,such as a bacterial cell or a fungal cell, using methods known in theart.

The nucleotide sequences of the present invention may be introduced intoa wide variety of prokaryotic and eukaryotic microorganism hosts toproduce the stabilized dsRNA or siRNA molecules. The term“microorganism” includes prokaryotic and eukaryotic microbial speciessuch as bacteria, fungi and algae. Fungi include yeasts and filamentousfungi, among others. Illustrative prokaryotes, both Gram-negative andGram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia,Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such asRhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia,Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae;Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae,Actinomycetales, and Nitrobacteraceae. Among eukaryotes are fungi, suchas Phycomycetes and Ascomycetes, which includes yeast, such asSaccharomyces and Schizosaccharomyces; and Basidiomycetes, such asRhodotorula, Aureobasidium, Sporobolomyces, and the like.

For the purpose of plant protection against insects, a large number ofmicroorganisms known to inhabit the phylloplane (the surface of theplant leaves) and/or the rhizosphere (the soil surrounding plant roots)of a wide variety of important crops may also be desirable host cellsfor manipulation, propagation, storage, delivery and/or mutagenesis ofthe disclosed recombinant constructs. These microorganisms includebacteria, algae, and fungi. Of particular interest are microorganisms,such as bacteria, e.g., genera Bacillus (including the species andsubspecies B. thuringiensis kurstaki HD-1, B. thuringiensis kurstakiHD-73, B. thuringiensis sotto, B. thuringiensis berliner, B.thuringiensis thuringiensis, B. thuringiensis tolworthi, B.thuringiensis dendrolimus, B. thuringiensis alesti, B. thuringiensisgalleriae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B.thuringiensis entomocidus, B. thuringiensis tenebrionis and B.thuringiensis san diego); Pseudomonas, Erwinia, Serratia, Klebsiella,Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., generaSaccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula,and Aureobasidium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobactersphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligeneseutrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans.

D. Transgenic Plants

The present invention provides seeds and plants having one or moretransgenic event. Combinations of events are referred to as “stacked”transgenic events. These stacked transgenic events can be events thatare directed at the same target pest, or they can be directed atdifferent target pests. In one embodiment, a seed having the ability toexpress a nucleic acid provided herein also has the ability to expressat least one other insecticidal agent, including, but not limited to, anRNA molecule the sequence of which is derived from the sequence of anRNA expressed in a target pest and that forms a double stranded RNAstructure upon expressing in the seed or cells of a plant grown from theseed, wherein the ingestion of one or more cells of the plant by thetarget pest results in the suppression of expression of the RNA in thecells of the target pest.

In certain embodiments, a seed having the ability to express a dsRNA thesequence of which is derived from a target pest also has a transgenicevent that provides herbicide tolerance. One beneficial example of aherbicide tolerance gene provides resistance to glyphosate,N-(phosphonomethyl) glycine, including the isopropylamine salt form ofsuch herbicide.

In the present method, combination of expression of an insecticidalamount of a dsRNA within the cells of a transgenic seed or plant grownfrom the seed coupled with treatment of the seed or plant with certainchemical or protein pesticides may be used to provide unexpectedsynergistic advantages, including unexpectedly superior efficacy forprotection against damage to the resulting transgenic plant by thetarget pest. In particular embodiments, treatment of a transgenic seedthat is capable of expressing certain constructs that form dsRNAmolecules, the sequence of which are derived from one or more sequencesexpressed in a corn rootworm, with from about 100 gm to about 400 gm ofpesticide per 100 kg of seed provides unexpectedly superior protectionagainst corn rootworm. In addition, it is believed that suchcombinations are also effective to protect the emergent plants againstpredation by other pests. The seeds of the present invention may also beused to decrease the cost of pesticide use, because less pesticide canbe used to obtain a required amount of protection than when such methodsare not used. Moreover, because less pesticide is used and because it isapplied prior to planting and without a separate field application, itis believed that the subject method is therefore safer to the operatorand to the environment, and is potentially less expensive thanconventional methods.

By “synergistic” it is meant to include the synergistic effects of thecombination on the pesticidal activity (or efficacy) of the combinationof the transgenic event and the pesticide. However, it is not intendedthat such synergistic effects be limited to the pesticidal activity, butthat they should also include such unexpected advantages as increasedscope of activity, advantageous activity profile as related to type andamount of damage reduction, decreased cost of pesticide and application,decreased pesticide distribution in the environment, decreased pesticideexposure of personnel who produce, handle and plant corn seeds, andother advantages known to those skilled in the art.

Pesticides and insecticides that are useful in compositions incombination with the methods and compositions of the present invention,including as seed treatments and coatings as well as methods for usingsuch compositions can be found, for example, in U.S. Pat. No. 6,551,962,the entirety of which is incorporated herein by reference.

Although it is believed that the seed treatments can be applied to atransgenic seed in any physiological state, it may be preferred that theseed be in a sufficiently durable state that it incurs no damage duringthe treatment process. Typically, the seed would be a seed that had beenharvested from the field; removed from the transgenic plant; andseparated from any other non-seed plant material. The seed wouldpreferably also be biologically stable to the extent that thetreatment-would cause no biological damage to the seed. In oneembodiment, for example, the treatment can be applied to seed corn thathas been harvested, cleaned and dried to a moisture content below about15% by weight. In an alternative embodiment, the seed can be one thathas been dried and then primed with water and/or another material andthen re-dried before or during the treatment with the pesticide. Withinthe limitations described, it is believed that the treatment can beapplied to the seed at any time between harvest of the seed and sowingof the seed. As used herein, the term “unsown seed” is meant to includeseed at any period between the harvest of the seed and the sowing of theseed in the ground for the purpose of germination and growth of theplant. When it is said that unsown seed is “treated” with the pesticide,such treatment is not meant to include those practices in which thepesticide is applied to the soil, rather than to the seed. For example,such treatments as the application of the pesticide in bands, “T”-bands,or in-furrow, at the same time as the seed is sowed are not consideredto be included in the present invention.

The pesticide, or combination of pesticides, can be applied “neat”, thatis, without any diluting or additional components present. However, thepesticide is typically applied to the seeds in the form of a pesticideformulation. This formulation may contain one or more other desirablecomponents including but not limited to liquid diluents, binders toserve as a matrix for the pesticide, fillers for protecting the seedsduring stress conditions, and plasticizers to improve flexibility,adhesion and/or spreadability of the coating.

The subject pesticides can be applied to a seed as a component of a seedcoating. Seed coating methods and compositions that are known in the artare useful when they are modified by the addition of one of theembodiments of the combination of pesticides of the present invention.Such coating methods and apparatus for their application are disclosedin, for example, U.S. Pat. Nos. 5,918,413, 5,891,246, 5,554,445,5,389,399, 5,107,787, 5,080,925, 4,759,945 and 4,465,017. Seed coatingcompositions are disclosed, for example, in U.S. Pat. Nos. 5,939,356,5,882,713, 5,876,739, 5,849,320, 5,834,447, 5,791,084, 5,661,103,5,622,003, 5,580,544, 5,328,942, 5,300,127, 4,735,015, 4,634,587,4,383,391, 4,372,080, 4,339,456, 4,272,417 and 4,245,432, among others.

The pesticides that are useful in the coating are those pesticides thatare described herein. The amount of pesticide that is used for thetreatment of the seed will vary depending upon the type of seed and thetype of active ingredients, but the treatment will comprise contactingthe seeds with an amount of the combination of pesticides that ispesticidally effective. When insects are the target pest, that amountwill be an amount of the insecticide that is insecticidally effective.As used herein, an insecticidally effective amount means that amount ofinsecticide that will kill insect pests in the larvae or pupal state ofgrowth, or will consistently reduce or retard the amount of damageproduced by insect pests.

In general, the amount of pesticide that is applied to the seed in thetreatment will range from about 10 gm to about 2000 gm of the activeingredient of the pesticide per 100 kg of the weight of the seed.Preferably, the amount of pesticide will be within the range of about 50gm to about 1000 gm active per 100 kg of seed, more preferably withinthe range of about 100 gm to about 600 gm active per 100 kg of seed, andeven more preferably within the range of about 200 gm to about 500 gm ofactive per 100 kg of seed weight. Alternatively, it has been found to bepreferred that the amount of the pesticide be over about 60 gm of theactive ingredient of the pesticide per 100 kg of the seed, and morepreferably over about 80 gm per 100 kg of seed.

The pesticides that are used in the treatment must not inhibitgermination of the seed and should be efficacious in protecting the seedand/or the plant during that time in the target insect's life cycle inwhich it causes injury to the seed or plant. In general, the coatingwill be efficacious for approximately 0 to 120 days after sowing. Thepesticides of the subject invention can be applied to the seed in theform of a coating.

Benefits provided by the present invention may include, but are notlimited to: the ease of introducing dsRNA into the insect cells, the lowconcentration of dsRNA which can be used, the stability of dsRNA, andthe effectiveness of the inhibition. The ability to use a lowconcentration of a stabilized dsRNA avoids several disadvantages ofanti-sense interference. The present invention is not limited to invitro use or to specific sequence compositions, to a particular set oftarget genes, a particular portion of the target gene's nucleotidesequence, or a particular transgene or to a particular delivery method,as opposed to the some of the available techniques known in the art,such as antisense and co-suppression. Furthermore, genetic manipulationbecomes possible in organisms that are not classical genetic models.

In practicing the present invention, selections can be carried out toensure that the presence of the nucleotide sequences that aretranscribed from the recombinant construct are not harmful to non-pestcells. This can be achieved by targeting genes that exhibit a low degreeof sequence identity with corresponding genes in a plant or a vertebrateanimal. Preferably the degree of the sequence identity is less thanapproximately 80%. More preferably the degree of the sequence identityis less than approximately 70%. Most preferably the degree of thesequence identity is less than approximately 60%.

In addition to direct transformation of a plant with a recombinant DNAconstruct, transgenic plants can be prepared by crossing a first planthaving a recombinant DNA construct with a second plant lacking theconstruct. For example, recombinant DNA for gene suppression can beintroduced into first plant line that is amenable to transformation toproduce a transgenic plant that can be crossed with a second plant lineto introgress the recombinant DNA for gene suppression into the secondplant line.

The present invention can be, in practice, combined with other insectcontrol traits in a plant to achieve desired traits for enhanced controlof insect infestation. Combining insect control traits that employdistinct modes-of-action can provide insect-protected transgenic plantswith superior durability over plants harboring a single insect controltrait because of the reduced probability that resistance will develop inthe field.

The mechanism of insecticidal activity of B. thuringiensis crystalproteins has been studied extensively in the past decade. It has beenshown that the crystal proteins are toxic to the larval form of theinsect only after ingestion of the protein. In lepidopteran larvae, analkaline pH and proteolytic enzymes in the insect mid-gut solubilize theproteins, thereby allowing the release of components that are toxic tothe insect. These toxic components disrupt the mid-gut cells, cause theinsect to cease feeding, and, eventually, bring about insect death. Forthis reason, B. thuringiensis toxins have proven themselves to beeffective and environmentally safe insecticides in dealing with variousinsect pests. Coleopteran and hemipteran insects, and likely dipteran,lygus and other piercing and sucking insects exhibit a gut pH that isslightly acidic, and so the Bt toxins that are effective againstlepidopteran larvae are ineffective against these pests. The slightlyacidic pH of the gut of these insects is also believed to be morehospitable to the compositions of the present invention, and withoutintending to be limited to a particular theory, it is likely that thealkaline pH of the gut of lepidopteran larvae is a contributing reasonthat prior attempts to exhibit dsRNA efficacy has failed (Fire et al.U.S. Pat. No. 6,506,559; Mesa et al. Patent Publication No.US2003/0150017; Rajagopal et al., 2002; Tabara et al., 1998). It isbelieved therefore that the dsRNA methods disclosed herein should bepreferentially used in compositions and in plants to controlcoleopteran, dipteran, hemipteran, lygus, and piercing and suckinginsects. The methods and compositions set forth herein are particularlyuseful for targeting genes for suppression in insects exhibiting a gutpH of from about 4.5 to about 9.5, or from about 5.0 to about 9.0, orfrom about 5.5 to about 8.5, or from about 6.0 to about 8.0, or fromabout 6.5 to about 7.7, or from about 6.8 to about 7.6, or about 7.0.However, insects and other pest species that exhibit a gut pH of fromabout 7.5 to about 11.5, or from about 8.0 to about 11.0, or from about9.0 to about 10.0, such as lepidopteran insect larvae, are also intendedto be within the scope of the present invention. This is particularlytrue when a dsRNA specific for inhibiting a gene in a lepidopteranlarvae is provided in the diet of the larvae along with one or more Btproteins, that, with respect to the Bt protein would ordinarily be toxicto that lepidopteran larvae when provided at or above a threshold level.The presence of one or more Bt toxins toxic to the same insect specieswould effectively reduce the gut pH, providing a stable environment forthe double stranded RNA molecules to exert their effects in suppressinga target gene in the insect pest.

It is anticipated that the combination of certain stabilized dsRNAconstructs with one or more insect control protein genes will result insynergies that enhance the insect control phenotype of a transgenicplant. Insect bioassays employing artificial diet- or whole plant tissuecan be used to define dose-responses for larval mortality or growthinhibition using both dsRNAs and insect control proteins. One skilled inthe art can test mixtures of dsRNA molecules and insect control proteinsin bioassay to identify combinations of actives that are synergistic anddesirable for deployment in insect-protected plants (Tabashnik, 1992).Synergy in killing insect pests has been reported between differentinsect control proteins (for review, see Schnepf et al., 1998). It isanticipated that synergies will exist between certain dsRNAs and betweencertain dsRNAs and certain insect control proteins.

The invention also relates to commodity products containing one or moreof the sequences of the present invention, and produced from arecombinant plant or seed containing one or more of the nucleotidesequences of the present invention are specifically contemplated asembodiments of the present invention. A commodity product containing oneor more of the sequences of the present invention is intended toinclude, but not be limited to, meals, oils, crushed or whole grains orseeds of a plant, or any food product comprising any meal, oil, orcrushed or whole grain of a recombinant plant or seed containing one ormore of the sequences of the present invention. The detection of one ormore of the sequences of the present invention in one or more commodityor commodity products contemplated herein is defacto evidence that thecommodity or commodity product is composed of a transgenic plantdesigned to express one or more of the nucleotides sequences of thepresent invention for the purpose of controlling insect infestationusing dsRNA mediated gene suppression methods.

D. Obtaining Nucleic acids

The present invention provides a method for obtaining a nucleic acidcomprising a nucleotide sequence for producing a dsRNA or siRNA. In oneembodiment, such a method comprises: (a) probing a cDNA or gDNA librarywith a hybridization probe comprising all or a portion of a nucleotidesequence or a homolog thereof from a targeted insect; (b) identifying aDNA clone that hybridizes with the hybridization probe; (c) isolatingthe DNA clone identified in step (b); and (d) sequencing the cDNA orgDNA fragment that comprises the clone isolated in step (c) wherein thesequenced nucleic acid molecule transcribes all or a substantial portionof the RNA nucleotide acid sequence or a homolog thereof.

In another embodiment, a method of the present invention for obtaining anucleic acid fragment comprising a nucleotide sequence for producing asubstantial portion of a dsRNA or siRNA comprises: (a) synthesizingfirst and a second oligonucleotide primers corresponding to a portion ofone of the nucleotide sequences from a targeted insect; and (b)amplifying a cDNA or gDNA template in a cloning vector using the firstand second oligonucleotide primers of step (a) wherein the amplifiednucleic acid molecule transcribes a substantial portion of a dsRNA orsiRNA of the present invention.

In practicing the present invention, a target gene may be derived from acorn rootworm (CRW), such as a WCR or a SCR, or any insect species thatcauses damage to the crop plants and subsequent yield losses. It iscontemplated that several criteria may be employed in the selection ofpreferred target genes. The gene is one whose protein product has arapid turnover rate, so that dsRNA inhibition will result in a rapiddecrease in protein levels. In certain embodiments it is advantageous toselect a gene for which a small drop in expression level results indeleterious effects for the insect. If it is desired to target a broadrange of insect species a gene is selected that is highly conservedacross these species. Conversely, for the purpose of conferringspecificity, in certain embodiments of the invention, a gene is selectedthat contains regions that are poorly conserved between individualinsect species, or between insects and other organisms. In certainembodiments it may be desirable to select a gene that has no knownhomologs in other organisms.

As used herein, the term “derived from” refers to a specified nucleotidesequence that may be obtained from a particular specified source orspecies, albeit not necessarily directly from that specified source orspecies.

In one embodiment, a gene is selected that is expressed in the insectgut. Targeting genes expressed in the gut avoids the requirement for thedsRNA to spread within the insect. Target genes for use in the presentinvention may include, for example, those that share substantialhomologies to the nucleotide sequences of known gut-expressed genes thatencode protein components of the vacuolar and plasma membrane protonV-ATPase (Dow et al., 1997; Dow, 1999). This protein complex is the soleenergizer of epithelial ion transport and is responsible foralkalinization of the midgut lumen. The V-ATPase is also expressed inthe Malpighian tubule, an outgrowth of the insect hindgut that functionsin fluid balance and detoxification of foreign compounds in a manneranalogous to a kidney organ of a mammal. In another embodiment, theV-ATPase may be Vha68-2, or a homolog or ortholog thereof (e.g. as foundin SEQ ID NO:821).

In another embodiment, a gene is selected that is essentially involvedin the growth, development, and reproduction of an insect. Exemplarygenes include but are not limited to a CHD3 gene, a β-tubulin gene, anda gene encoding a protein predicted to be involved in transport. TheCHD3 gene in Drosophila melanogaster encodes a protein withATP-dependent DNA helicase activity that is involved in chromatinassembly/disassembly in the nucleus. Similar sequences have been foundin diverse organisms such as Arabidopsis thaliana, Caenorhabditiselegans, and Saccharomyces cerevisiae. The beta-tubulin gene familyencodes microtubule-associated proteins that are a constituent of thecellular cytoskeleton. Related sequences are found in such diverseorganisms as C. elegans, and Manduca sexta. Proteins predicted to besubunits of the endosomal sorting complex required for transport(ESCRT)-III (Babst et al., 2002), e.g. Dv49, are found in diverseorganisms including mammals, yeast, and insects such as D. virgifera.Another transport-related protein is the β′-coatomer protein,abbreviated as β′Cop, that encodes a product involved in retrograde(Golgi to ER) transport. Similar or predicted sequences have beenidentified in C. elegans and D. virgifera, e.g. Dv248 (SEQ ID NO:.

Other target genes for use in the present invention may include, forexample, those that play important roles in the viability, growth,development, reproduction and infectivity. These target genes may be oneof the house keeping genes, transcription factors and insect specificgenes or lethal knockout mutations in Drosophila. The target genes foruse in the present invention may also be those that are from otherorganisms, e.g., from a nematode (e.g., C. elegans). Additionally, thenucleotide sequences for use in the present invention may also bederived from plant, viral, bacterial or fungal genes whose functionshave been established from literature and the nucleotide sequences ofwhich share substantial similarity with the target genes in the genomeof an insect. According to one aspect of the present invention for WCRcontrol, the target sequences may essentially be derived from thetargeted WCR insect. Some of the exemplary target sequences from cDNAlibrary from WCR that encode D. virgifera proteins or fragments thereofwhich are homologues of known proteins may be found in the SequenceListing. Nucleic acid molecules from D. virgifera encoding homologs ofknown proteins are known (Andersen et al., U.S. patent application Ser.No. 10/205,189).

For the purpose of the present invention, the dsRNA or siRNA moleculesmay be obtained from the CRW by polymerase chain (PCR™) amplification ofa target CRW gene sequences derived from a corn rootworm gDNA or cDNAlibrary or portions thereof. The WCR larvae may be prepared usingmethods known to the ordinary skilled in the art and DNA/RNA may beextracted. Larvae with various sizes ranging from 1st instars tofully-grown CRWs may be used for the purpose of the present inventionfor DNA/RNA extraction. Genomic DNA or cDNA libraries generated from WCRmay be used for PCR™ amplification for production of the dsRNA or siRNA.

The target genes may be then be PCR™ amplified and sequenced using themethods readily available in the art. One skilled in the art may be ableto modify the PCR™ conditions to ensure optimal PCR™ product formation.The confirmed PCR™ product may be used as a template for in vitrotranscription to generate sense and antisense RNA with the includedminimal promoters.

The present inventors contemplate that nucleic acid sequences identifiedand isolated from any insect species in the insect kingdom may be usedin the present invention for control of WCR and another targetedinsects. In one aspect of the present invention, the nucleic acid may bederived from a coleopteran species. Specifically, the nucleic acid maybe derived from leaf beetles belonging to the genus Diabrotica(Coleoptera, Chrysomelidae) and more specifically the nucleic acidmolecules of the present invention may be derived from species in thevirgifera group. Most specifically, the nucleic acid molecules of thepresent invention may be derived from Diabrotica virgifera virgiferaLeConte that is normally referred to as WCR. The isolated nucleic acidsmay be useful, for example, in identifying a target gene and inconstructing a recombinant vector that produce stabilized dsRNAs orsiRNAs of the present invention for protecting plants from WCR insectinfestations.

Therefore, in one embodiment, the present invention comprises isolatedand purified nucleotide sequences from WCR or Lygus that may be used asthe insect control agents. The isolated and purified nucleotidesequences may comprise those as set forth in the sequence listing.

The nucleic acids from WCR or other insects that may be used in thepresent invention may also comprise isolated and substantially purifiedUnigenes and EST nucleic acid molecules or nucleic acid fragmentmolecules thereof. EST nucleic acid molecules may encode significantportions of, or indeed most of, the polypeptides. Alternatively, thefragments may comprise smaller oligonucleotides having from about 15 toabout 250 nucleotide residues, and more preferably, about 15 to about 30nucleotide residues. Alternatively, the nucleic acid molecules for usein the present invention may be from cDNA libraries from WCR, or fromany other coleopteran pest species.

Nucleic acid molecules and fragments thereof from WCR, or othercoleopteran pest species may be employed to obtain other nucleic acidmolecules from other species for use in the present invention to producedesired dsRNA and siRNA molecules. Such nucleic acid molecules includethe nucleic acid molecules that encode the complete coding sequence of aprotein and promoters and flanking sequences of such molecules. Inaddition, such nucleic acid molecules include nucleic acid moleculesthat encode for gene family members. Such molecules can be readilyobtained by using the above-described nucleic acid molecules orfragments thereof to screen, for instance, cDNA or gDNA librariesobtained from D. v. virgifera or other coleopterans, or from Lygushesperus. Methods for forming such libraries are well known in the art.

As used herein, the phrase “coding sequence”, “structural nucleotidesequence” or “structural nucleic acid molecule” refers to a nucleotidesequence that is translated into a polypeptide, usually via mRNA, whenplaced under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a translation startcodon at the 5′-terminus and a translation stop codon at the3′-terminus. A coding sequence can include, but is not limited to,genomic DNA, cDNA, EST and recombinant nucleotide sequences.

The term “recombinant DNA” or “recombinant nucleotide sequence” refersto DNA that contains a genetically engineered modification throughmanipulation via mutagenesis, restriction enzymes, and the like.

For many of the insects that are potential targets for control by thepresent invention, there may be limited information regarding thesequences of most genes or the phenotype resulting from mutation ofparticular genes. Therefore, the present inventors contemplate thatselection of appropriate genes from insect pests for use in the presentinvention may be accomplished through use of information available fromstudy of the corresponding genes in a model organism such in Drosophila,in some other insect species, or even in a nematode species, in a fungalspecies, in a plant species, in which the genes have been characterized.In some cases it will be possible to obtain the sequence of acorresponding gene from a target insect by searching databases such asGenBank using either the name of the gene or the sequence from, forexample, Drosophila, another insect, a nematode, a fungus, or a plantfrom which the gene has been cloned. Once the sequence is obtained, PCR™may be used to amplify an appropriately selected segment of the gene inthe insect for use in the present invention.

In order to obtain a DNA segment from the corresponding gene in aninsect species, PCR™ primers may be designed based on the sequence asfound in WCR or other insects from which the gene has been cloned. Theprimers are designed to amplify a DNA segment of sufficient length foruse in the present invention. DNA (either genomic DNA or cDNA) isprepared from the insect species, and the PCR™ primers are used toamplify the DNA segment. Amplification conditions are selected so thatamplification will occur even if the primers do not exactly match thetarget sequence. Alternately, the gene (or a portion thereof) may becloned from a gDNA or cDNA library prepared from the insect pestspecies, using the WCR gene or another known insect gene as a probe.Techniques for performing PCR™ and cloning from libraries are known.Further details of the process by which DNA segments from target insectpest species may be isolated based on the sequence of genes previouslycloned from WCR or other insect species are provided in the Examples.One of ordinary skill in the art will recognize that a variety oftechniques may be used to isolate gene segments from insect pest speciesthat correspond to genes previously isolated from other species.

When an insect is the target pest for the present invention, such pestsinclude but are not limited to: from the order Lepidoptera, for example,

Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabamaargillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp,Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella,Carposina nipponensis, Chilo spp., Choristoneura spp., Clysiaambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp.,Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydiaspp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp.,Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.,Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis,Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella,Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp.,Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp.,Ostrinia Nubilalis, Pammene spp., Pandemis spp., Panolis flammea,Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pierisspp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp.,Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp.,Tortrix spp., Trichoplusia ni and Yponomeuta spp.;

from the order Coleoptera, for example,

Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp.,Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrusspp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinusspp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeidae,Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. andTrogoderma spp.;

from the order Orthoptera, for example,

Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae,Locusta spp., Periplaneta ssp., and Schistocerca spp.;

from the order Isoptera, for example,

Reticulitemes ssp;

from the order Psocoptera, for example,

Liposcelis spp.;

from the order Anoplura, for example,

Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. andPhylloxera spp.;

from the order Mallophaga, for example,

Damalinea spp. and Trichodectes spp.;

from the order Thysanoptera, for example,

Franklinella spp., Hercinothrips spp., Taeniothrips spp., Thrips palmi,Thrips tabaci and Scirtothrips aurantii;

from the order Heteroptera, for example,

Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp.,Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodniusspp., Sahlbergella singularis, Scotinophara spp., Triatoma spp., Miridaefamily spp. such as Lygus hesperus and Lygus lineoloris, Lygaeidaefamily spp. such as Blissus leucopterus, and Pentatomidae family spp.;

from the order Homoptera, for example,

Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp.,Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplasterspp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccushesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,Gascardia spp., Laodelphax spp., Lacanium corni, Lepidosaphes spp.,Macrosiphus spp., Myzus spp., Nehotettix spp., Nilaparvata spp.,Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp.,Pseudococcus spp., Psylla ssp., Pulvinaria aethiopica, Quadraspidiotusspp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphisspp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae andUnaspis citri;

from the order Hymenoptera, for example,

Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpiniapolytoma, Hoplocampa spp., Lasius sppp., Monomorium pharaonis,Neodiprion spp, Solenopsis spp. and Vespa ssp.;

from the order Diptera, for example,

Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphoraerythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebraspp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilusspp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomysa spp.,Lucilia spp., Melanagromyza spp., Musca ssp., Oestrus spp., Orseoliaspp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletispomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. andTipula spp.,

from the order Siphonaptera, for example,

Ceratophyllus spp. und Xenopsylla cheopis and

from the order Thysanura, for example,

Lepisma saccharina.

It has been found that the present invention is particularly effectivewhen the insect pest is a Diabrotica spp., and especially when the pestis Diabrotica virgifera virgifera (Western Corn Rootworm, WCR),Diabrotica barberi (Northern Corn Rootworm, NCR), Diabrotica virgiferazea (Mexican Corn Rootworm, MCR), Diabrotica balteata (Brazilian CornRootworm (BZR) or Brazilian Corn Rootworm complex (BCR) consisting ofDiabrotica viridula and Diabrotica speciosa), or Diabroticaundecimpunctata howardi (Southern Corn Rootworm, SCR).

EXAMPLES

The inventors herein have identified means for controlling coleopteranpest infestation by providing a double stranded ribonucleic acidmolecules in the diet of pests. Surprisingly, the inventors havediscovered double stranded ribonucleic acid molecules that function uponingestion by the pest to inhibit a biological function in the pest,resulting in one or more of the following attributes: reduction infeeding by the pest, reduction in viability of the pest, death of thepest, inhibition of differentiation and development of the pest, absenceof or reduced capacity for sexual reproduction by the pest, muscleformation, juvenile hormone formation, juvenile hormone regulation, ionregulation and transport, maintenance of cell membrane potential, aminoacid biosynthesis, amino acid degradation, sperm formation, pheromonesynthesis, pheromone sensing, antennae formation, wing formation, legformation, development and differentiation, egg formation, larvalmaturation, digestive enzyme formation, haemolymph synthesis, haemolymphmaintenance, neurotransmission, cell division, energy metabolism,respiration, apoptosis, and any component of a eukaryotic cells'cytoskeletal structure, such as, for example, actins and tubulins. Anyone or any combination of these attributes can result in an effectiveinhibition of pest infestation, and in the case of a plant pest,inhibition of plant infestation. For example, when used as a dietcomposition containing a pest inhibitory sufficient amount of one ormore double stranded ribonucleic acid molecules provided topically to aplant, as a seed treatment, as a soil application around a plant, orwhen produced by a plant from a recombinant DNA molecule present withinthe cells of a plant, plant pest infestation is unexpectedlydramatically reduced. The Examples set forth herein below areillustrative of the invention when applied to a single pest. However,the skilled artisan will recognize that the methods, formulae, and ideaspresented in the Examples are not intended to be limiting, and areapplicable to all coleopteran pest species that can consume food sourcesthat can be formulated to contain a sufficient amount of a pestinhibitory agent consisting at least of one or more double stranded RNAmolecules exemplified herein intended to suppress some essential featureabout or function within the pest.

Example 1 Identification of Target Nucleotide Sequences For Preparationof dsRNA Useful for Controlling Corn Rootworms

Corn rootworm cDNA libraries (LIB149, LIB 150, LIB33027, LIB3373) wereconstructed from whole larvae, pupae and from dissected midgut sections,and nucleotide sequence information was obtained (see Andersen et al.,U.S. patent application Ser. No. 10/205,189 filed Jul. 24, 2002,incorporated herein specifically by reference in its entirety). Inaddition, cDNA libraries were constructed from whole larvae at differentdevelopmental stages and at different times within each developmentalstage in order to maximize the number of different EST sequences fromthe Diabrotica species. Libraries LIB5444 and LIB5462 were constructedrespectively from mRNA pools obtained from first (1 gram) and third (2.9grams) instar Western Corn Rootworm larvae. Harvested insects wererapidly frozen by insertion into liquid nitrogen. The insects wereground in a mortar and pestle maintained at or below −20° C. by chillingon dry ice and/or with the addition of liquid nitrogen to the mortaruntil the tissue was ground into a fine powder. RNA was extracted usingTRIzol® reagent (Invitrogen) according to the manufacturer'sinstructions. Poly A+ RNA was isolated from the total RNA prep usingDYNABEADS Oligo dT (Invitrogen) following the manufacturer'sinstructions. A cDNA library was constructed from the Poly A+ RNA usingthe SuperScript™ Plasmid System (Invitrogen). cDNA was size fractionatedusing chromatography. The fourth and fifth fractions were collected andligated into the pSPORT1 vector (Life Technologies Inc., GaithersburgMd.) between the SalI and NotI restriction endonucleases recognitionsites, and transformed into E. coli DH10B electro-competent cells byelectroporation. The first instar larvae library yielded about 420,000colony-forming units. The third instar larvae library yielded about2.78×10⁶ colony forming units. Colonies from LIB149, LIB150 were washedfrom the plates, mixed to uniformity by vortexing briefly, and pooledinto Tris-EDTA buffer. Half of the wash was brought to 10% glycerol,aliquoted into cryovials, and stored at −70° C. The other half was usedto produce plasmid DNA using a Quiagen midi-prep purification column, orits equivalent. Purified plasmid DNA was aliquoted to microcentrifugetubes and stored at −20° C.

Colonies from the Diabrotica virgifera cDNA libraries LIB5444 andLIB5462 were amplified individually in a high viscosity medium.Approximately 200,000 colony-forming units from LIB5444 and 600,000colony-forming units from LIB5462 were mixed on a stir plate separatelyin 500 ml LB medium containing 0.3% SeaPrep Agarose® and 50 mg/lcarbenecillin at 37° C. and then rapidly cooled in a water/ice bath for1 hour allowing uniform suspension of the bacterial colonies. Theinoculated libraries were then grown at 30° C. for 42 hours. Afterincubation, the cells were mixed for 5 minutes on a stir plate. Themedium was then transferred to two 250 ml centrifuge bottles. Thebacterial cells were pelleted at 10,000×g for 10 minutes. The medium wasremoved from the bottles and the cells were resuspended in a total of 20ml of LB medium with 50 mg/l carbenecillin. Dimethyl sulfoxide was addedto 10% to preserve the cells in freezing. Both libraries were amplifiedto a final titer of 10⁸ colony-forming units per milliliter. Samples ofthe Diabrotica virgifera cDNA libraries LIB5444 and LIB5462 werecombined and adjusted to a DNA concentration of about 1.25 microgramsper microliter in sterile distilled and deionized water and aliquotedinto twenty five cryovials, each cryovial containing about 8.75micrograms of DNA. These samples were deposited by theapplicant(s)/inventors with the American Type Culture Collection (ATCC)located at 10801 University Boulevard, Manassas, Va., USA ZIP 20110-2209on Jun. 10, 2004 and referred to as LIB5444/62. The ATCC provided theApplicant with a deposit receipt, assigning the ATCC Deposit AccessionNo. PTA-6072.

Corn rootworm high molecular weight cDNA libraries, i.e., LIB5496 andLIB5498, were prepared essentially as described above for the productionof corn rootworm cDNA libraries. Libraries LIB5496 and LIB5498 wereconstructed respectively from mRNA pools obtained from first (1 gram)and second and third (1 gram) instar Western Corn Rootworm larvae.Briefly, insects were quickly frozen in liquid nitrogen. The frozeninsects were reduced to a fine powder by grinding in a mortar andpestle. RNA was extracted using TRIzol® reagent (Invitrogen) followingthe manufacturer's instructions. Poly A+ RNA was isolated from the totalRNA prep using DYNABEADS Oligo dT (Invitrogen). A high molecular weightcDNA library was made from 20 micrograms of Poly A+ RNA using theSuperScript™ Plasmid System (Invitrogen). The cDNA was size fractionatedon a 1% agarose gel in TAE, and cDNA between the range of 1 Kb to 10 Kbwas collected and ligated into the pSPORT1 vector in between the Sal1and Not1 restriction sites and transformed into E. coli DH10Belectro-competent cells by electroporation. LIB5496 yielded a totaltiter of about 3.5×10⁶ colony forming units. LIB5498 yielded a totaltiter of about 1.0×10⁶ colony forming units. Colonies from the cornrootworm high molecular weight cDNA libraries LIB5496 and LIB5498 wereamplified individually in a high viscosity medium. Approximately 600,000colony-forming units from LIB5496 and LIB5498 were mixed on a stir plateseparately in 500 ml LB medium containing 0.3% SeaPrep Agarose® and 50mg/l carbenecillin at 37° C. and then rapidly cooled in a water/ice bathfor 1 hour allowing uniform suspension of the bacterial colonies. Thelibraries were then grown at 30° C. for 42 hours. After incubation, thecells were mixed for 5 minutes on a stir plate. The medium was thentransferred to two 250 mL centrifuge bottles. The bacterial cells werepelleted at 10,000×g for 10 minutes. The medium was removed from thebottles and the cells were resuspended in a total of 20 mL of LB mediumwith 50 mg/L carbenecillin. Dimethyl sulfoxide was added to 10% topreserve the cells in freezing. Both libraries were amplified to a finaltiter of 10⁸ colony-forming units per milliliter. Inserted cDNA sequenceinformation was obtained from the corn rootworm species-specific plasmidlibraries.

The Andersen et al. rootworm libraries together with additionalsequences from the libraries LIB5444 and LIB5462 initially producedabout 18,415 individual EST sequences consisting of approximately1.0×10⁷ nucleotide residues. The average length of an EST sequence wasabout 586 nucleotide residues. These EST sequences were subjected tobioinformatics algorithms that resulted in the assembly of contigsequences referred to herein as UNIGENE sequences, and individual ESTsequences that could not be compiled by overlap identity with other ESTsequences, referred to herein as singletons. The LIB5444 and LIB5462libraries were then sequenced much deeper, resulting in additionalindividual EST sequences. EST sequences obtained from libraries, i.e.,LIB149, LIB150, LIB3027, LIB3373, LIB5444, LIB5462, LIB5496 and LIB5503were selected for further investigation in feeding bioassays as setforth below and the corresponding sequences are given in the sequencelisting.

The EST sequences isolated from CRW cDNA libraries were assembled, wherepossible, into UNIGENE sets and these assembled Unigene sequences areincluded in the sequence listing. A UNIGENE is a gene-oriented clusterformed from the overlap of individual EST sequences within regions ofsequence identity to form a larger sequence. Pontius et al. (2003). Eachnucleotide sequence as set forth in the sequence listing was analyzed toidentify the presence of open reading frames. Amino acid sequenceinformation deduced from open reading frames was compared to known aminoacid sequence information available in public databases in order todeduce the extent of amino acid sequence identity or similarity to thoseknown amino acid sequences. Biological function, if any, associated withknown amino acid sequences in public databases was annotated to theamino acid sequences deduced from the cDNA library nucleotide sequenceinformation. Annotations provided information that was suggestive of thefunction of a protein that may be expressed from a particular gene thatgave rise to a particular cDNA sequence, but was not outcomedeterminative. Based on the suggestive annotation information, certaincDNA sequences were characterized as those that encoded a protein thatwas likely involved in some biological function within corn rootwormcells that was either essential to life, or that was necessary forensuring health and vitality to a cell, or were likely to be involved incellular integrity, cell maintenance, reproductive capacity, and thelike.

Sequences selected for further investigation were used in theconstruction of double stranded RNA molecules for incorporation into CRWdiet. Thermal amplification primer pairs were designed based on cDNA andEST starting sequences to obtain sequences used in feeding assays.Primer pairs were constructed either as a pair of nucleotide sequences,each member of a primer pair exhibiting perfect complementarity eitherto a sense or to an antisense sequence. Some primer pair sequences wereconstructed so that each member of the pair exhibited a sequencecontaining a T7 phage RNA polymerase promoter at it's 5′ end. Preferablya higher fidelity first amplification reaction was carried out using afirst primer pair lacking a T7 promoter to generate a first ampliconusing CRW genomic DNA as template. Preferably, a cDNA or a mRNA sequenceis used as the template for the synthesis of a dsRNA molecule for use inthe present invention because eukaryotic genome sequences are recognizedin the art to contain sequences that are not present within the matureRNA molecule. A sample of the first amplicon generated from the higherfidelity first amplification reaction was then used as template in asecond thermal amplification reaction with a second primer paircontaining the T7 promoter sequence to produce a second amplicon thatcontained a T7 promoter at or embedded within the 5′ end of each strandof the second amplicon. The complete nucleotide sequence of the secondamplicon was obtained in both directions and compared to the nucleotidesequence as reported for the cDNA, and discrepancies between the twosequences, if any, were noted. Generally, sequences prepared usinggenome DNA as template were inconsistent with further use as dsRNAmolecules for use in achieving significant levels of suppression becauseof variations within the genome sequences that were not present withinthe mRNA or cDNA sequence.

An in vitro transcription reaction typically contained from about 1 toabout 2 micrograms of linearized DNA template, T7 polymerase reactionbuffer from a 10× concentrate, ribonucleotides ATP, CTP, GTP, and UTP ata final concentration of from between 50 and 100 mM each, and 1 unit ofT7 RNA polymerase enzyme. The RNA polymerase reaction was incubated atabout 37° C., depending on the optimal temperature of the RNA polymeraseused according to the manufacturers' instructions, for a period of timeranging from several minutes to several hours. Generally, reactions werecarried out for from about 2 to about 6 hours for transcription oftemplate sequences up to about 400 nucleotides in length, and for up to20 hours for transcription of template sequences greater than about 400nucleotides in length. Heating the reaction to 65° C. for fifteenminutes terminates RNA transcription. RNA transcription products wereprecipitated in ethanol, washed, air dried and resuspended in RNAse freewater to a concentration of about 1 microgram per microliter. Mosttranscripts which took advantage of the opposing T7 promoter strategyoutlined above produced double stranded RNA in the in vitrotranscription reaction, however, a higher yield of double stranded RNAwas obtained by heating the purified RNA to 65° C. and then slowlycooling to room temperature to ensure proper annealing of sense andantisense RNA segments. Double stranded RNA products were then incubatedwith DNAse I and RNAse at 37° C. for one hour to remove any DNA orsingle stranded RNA present in the mixture. Double stranded RNA productswere purified over a column according to the manufacturers' instructions(AMBION MEGAscript® RNAi KIT) and resuspended in 10 mM Tris-HCl buffer(pH 7.5) or RNAse free water to a concentration of between 0.1 and 1.0microgram per microliter.

The following nucleotide sequences were derived first as cDNA sequencesidentified in a corn rootworm mid-gut cDNA library (Andersen et al.,ibid), and were adapted for use in constructing double stranded RNAmolecules for use in testing the efficacy of inhibiting a biologicalfunction in a pest by feeding double stranded RNA molecules in the dietof the pest.

A. Chd3 Homologous Sequences

CHD genes have been identified in numerous eukaryotes, and thecorresponding proteins are proposed to function as chromatin-remodelingfactors. The term CHD is derived from the three domains of sequencehomology found in CHD proteins: a chromo (chromatin organizationmodifier) domain, a SNF2-related helicase/ATPase domain, and aDNA-binding domain, each of which is believed to confer a distinctchromatin-related activity. CHD proteins are separated into twocategories based on the presence or absence of another domain ofsequence homology, a PHD zinc finger domain, typically associated withchromatin related activity. CHD3 related proteins possess a PHD zincfinger domain, but CHD1 related proteins do not. Experimentalobservations have suggested a role for CHD3 proteins in repression oftranscription, and in some species have been shown to be a component ofa complex that contains histone deacetylase as a subunit. Deacetylationof histones is correlated with transcriptional inactivation, and so CHD3proteins have been implicated to function as repressors of transcriptionby virtue of being a component of a histone deacetylase complex (Ogas etal., 1999). Thus, suppression of CHD3 protein synthesis may be a usefultarget for double stranded RNA mediated inhibition of coleopteran pests.

B. Beta-Tubulin Homologous Sequences

Tubulin proteins are important structural components of many cellularstructures in all eukaryote cells and principally in the formation ofmicrotubules. Inhibition of microtubule formation in cells results incatastrophic effects including interference with the formation ofmitotic spindles, blockage of cell division, and the like. Therefore,suppression of tubulin protein formation may be a useful target fordouble stranded RNA mediated inhibition.

C. 40 kDa V-ATPase Homologous Sequences

Energy metabolism within subcellular organelles in eukaryotic systems isan essential function. Vacuolar ATP synthases are involved inmaintaining sufficient levels of ATP within vacuoles. Therefore,vacuolar ATP synthases may be a useful target for double stranded RNAmediated inhibition.

D. EF1α Homologous Sequences

Transcription elongation and transcription termination factors areessential to metabolism and may be advantageous targets for doublestranded RNA mediated inhibition.

E. 26S Proteasome Subunit p28 Homologous Sequences

The 26S proteasome is a large, ATP-dependent, multi-subunit proteasethat is highly conserved in all eukaryotes. It has a general function inthe selective removal of various short-lived proteins that are firstcovalently linked to ubiquitin and then subsequently degraded by the 26Sproteasome complex. The ubiquitin pathway plays an important role in thecontrol of the cell cycle by the specific degradation of a number ofregulatory proteins including mitotic cyclins and inhibitors ofcyclin-dependent kinases such as p27 of mammalian cells. Thus, thesuppression of 26S proteasome synthesis and suppression of synthesis ofits component subunits may be preferred targets for double stranded RNAmediated inhibition. (Smith et al., 1997).

F. Juvenile Hormone Epoxide Hydrolase Homologous Sequences

Insect juvenile hormone controls and regulates a variety of necessarybiological processes within the insect life cycle including but notnecessarily limited to metamorphosis, reproduction, and diapause.Juvenile hormone (JH) concentrations are required to peak at appropriatetimes within the haemolymph of the larval form of an insect pest, inparticular lepidopteran and coleopteran larvae, and then must bedegraded in order to terminate the effects of the hormone response.Enzymes involved in decreasing the concentration of juvenile hormone areeffective through two primary pathways of metabolic degradation. Onepathway involves juvenile hormone esterase (JHE), which hydrolyzes themethyl ester providing the corresponding acid. The second pathwayutilizes juvenile hormone epoxide hydrolase (JHEH) to achieve hydrolysisof the epoxide, resulting in formation of the diol. The contribution ofJHE in the degradation of JH is well understood and has been found to beinvariate between the lepidoptera and coleoptera species. Inhibition ofJH esterase has been associated with severe morphological changesincluding but not limited to larval wandering, deferred pupation, anddevelopment of malformed intermediates. In contrast, the contribution ofJHEH in JH metabolism is less well understood and had been shown to varybetween the species, but recent studies point to evidence that suggeststhat JHEH may be the primary route of metabolism of JH (Brandon J.Fetterolf, Doctoral Dissertation, North Carolina State University (Feb.10, 2002) Synthesis and Analysis of Mechanism Based Inhibitors ofJuvenile Hormone Epoxide Hydrolase from Insect Trichoplusia ni). In anyevent, disruption of either JH degradation pathway using genesuppression technology could be an effective target for double strandedRNA mediated pest inhibition.

G. Swelling Dependent Chloride Channel Protein Homologous Sequences

Swelling dependent chloride channel proteins have been postulated toplay a critical role in osmoregulation in eukaryotic animal cellsystems. Therefore, a nucleotide sequence exhibiting the ability toexpress an amino acid sequence that exhibits homology to previouslyidentified swelling dependent chloride channel proteins may be a usefultarget for RNA inhibition in a pest.

H. Glucose-6-phosphate 1-dehydrogenase Protein Homologous Sequences

Glucose-6-phosphate 1-dehydrogenase protein (G6PD) catalyzes theoxidation of glucose-6-phosphate to 6-phosphogluconate whileconcomitantly reducing the oxidized form of nicotinamide adeninedinucleotide phosphate (NADP+) to NADPH. NADPH is known in the art as arequired cofactor in many eukaryotic biosynthetic reactions, and isknown to maintain glutathione in its reduced form. Reduced glutathioneacts as a scavenger for dangerous oxidative metabolites in eukaryoticcells, and with the assistance of the enzyme glutathione peroxidase,convert harmful hydrogen peroxide to water (Beutler et al., 1991).Therefore, G6PD may be a preferable target for double stranded RNAmediated inhibition in a coleopteran pest.

I. Act42A Protein Homologous Sequences

Actin is a ubiquitous and highly conserved eukaryotic protein requiredfor cell motility and locomotion (Lovato et al., 2001). A number of CRWcDNA sequences were identified that were predicted to likely encodeactin or proteins exhibiting amino acid sequence structure related toactin proteins. Therefore, genes encoding actin homologues in a pestcell may be useful targets for double stranded RNA mediated inhibition.

J. ADP-ribosylation Factor 1 Homologous Sequences

ADP ribosylation factors have been demonstrated to be essential in cellfunction in that they play integral roles in the processes of DNA damagerepair, carcinogenesis, cell death, and genomic stability. Thus, itwould be useful to be able to selectively disrupt transcription ofADP-ribosylation factors in coleopteran pest species using doublestranded RNA mediated inhibition.

K. Transcription Factor IIB Protein Homologous Sequences

Transcription elongation and transcription termination factors, asindicated above, are essential to metabolism and may be advantageoustargets for double stranded RNA mediated inhibition to control oreliminate coleopteran pest infestation.

L. Chitinase Homologous Sequences

Chitin is a β(1→4)homopolymer of N-acetylglucosamine and is found ininsect exoskeletons. Chitin is formed from UDP-N-acetlglucosamine in areaction catalyzed by chitin synthase. Chitin is a structuralhomopolymer polysaccharide, and there are many enzymatic steps involvedin the construction of this highly branched and cross-linked structure.Chitin gives shape, rigidity and support to insects and provides ascaffolding to which internal organs such as muscles are attached.Chitin must also be degraded to some extent to mediate the stepsinvolved in the insect molting process. Therefore, it is believed thatdouble stranded RNA mediated inhibition of proteins in these pathwayswould be useful as a means for controlling coleopteran pest infestation.

M. Ubiquitin Conjugating Enzyme Homologous Sequences

The ubiquitin pathway plays an important role in the control of the cellcycle by the specific degradation of a number of regulatory proteinsincluding mitotic cyclins and inhibitors of cyclin-dependent kinasessuch as p27 of mammalian cells. Thus, genes encoding ubiquitin andassociated components may be a preferred target for double stranded RNAmediated inhibition. (Smith et al., 1997). The ubiquitin-dependentproteolytic pathway is one of the major routes by which intracellularproteins are selectively destroyed in eukaryotes. Conjugation ofubiquitin to substrate proteins is mediated by a remarkably diversearray of enzymes. Proteolytic targeting may also be regulated at stepsbetween ubiquitination of the substrate and its degradation to peptidesby the multi-subunit 26S protease. The complexity of the ubiquitinsystem suggests a central role for protein turnover in eukaryotic cellregulation, and implicates other proteins in the pathway includingubiquitin-activating enzyme, ubiquitin-conjugating enzyme,ubiquitin-protein ligase, and 26S proteasome subunit components.Therefore, it is believed that double stranded RNA mediated inhibitionof proteins in this pathway would be useful as a means for controllingcoleopteran pest infestation.

N. Glyceraldehyde-3-phosphate Dehydrogenase Homologous Sequences

The glycolytic pathway is an essential pathway in most organisms and isinvolved in the production of metabolic energy from the degradation ofglucose. One important enzyme in the second stage of the glycolyticpathway is glyceraldehyde-3-phosphate dehydrogenase (G3PDH), which, inthe presence of NAD+ and inorganic phosphate, catalyzes the oxidation of3-phospho-glyceraldehyde to 3-phosphoglyceroyl-phosphate along with theformation of NADH. The important component of this reaction is thestorage of energy through the formation of NADH. Genes encoding enzymesassociated with the glycolytic pathway, and particularly genes: encodingenzymes involved in the steps useful in formation of energy reserves maybe particularly useful targets for double stranded RNA mediatedinhibition in coleopteran pest species.

O. Ubiquitin B Homologous Sequences

As described above, the ubiquitin protein degradation pathway plays animportant role in the control of the cell cycle by the specificdegradation of a number of regulatory proteins including mitotic cyclinsand inhibitors of cyclin-dependent kinases such as p27 of mammaliancells. Thus, genes encoding ubiquitin and associated components may be apreferred target for double stranded RNA mediated inhibition. (Smith etal., 1997).

P. Juvenile Hormone Esterase Homologs

As indicated above, insect juvenile hormone controls and regulates avariety of necessary biological processes within the insect life cycleincluding but not necessarily limited to metamorphosis, reproduction,and diapause. Disruption of JH synthesis or degradation pathways usinggene suppression technology could be an effective target for doublestranded RNA mediated pest inhibition.

Q. Alpha Tubulin Homologous Sequences

Eukaryotic cells generally utilize cytoskeletal structural elements thatare important, not only as a mechanical scaffold, but also in sustainingthe shape of the cell. Semiflexible microfilaments make cells mobile,help them to divide in mitosis (cytokinesis) and, in vertebrate andinvertebrate animals, are responsible for muscular contraction. Therelatively stiff microtubules which are made up of alpha and betatubulin proteins play an important role in acting as a sort of highwayfor transport of vesicles and organelles and in the separation ofchromosomes during mitosis (karyokinesis). The flexible intermediatefilaments provide at least additional strength to the overall cellularstructure. The cytoskeleton is also known to be involved in signalingacross the cell cytoplasm. Taking these functions into account, it isbelieved that any disruption of the cytoskeleton or even subtle changesof its integrity may cause pathological consequences to a cell.

R. Transport Related Sequences

As indicated above, sorting and transport of various molecules within acell, including to appropriate organelles, as well as their secretion isan important physiological function. Such sorting pathways could includethose relying on the endosomal sorting complex required for transport(ESCRT), complexes I-III, among others. Thus, functions related totransport of polypeptides and other molecules may also be a preferredtarget for dsRNA-mediated inhibition.

Example 2 Insect Feeding Bioassays

Samples of double stranded RNA (dsRNA) were subjected to bioassay with aselected number of target pests. The dsRNA was prepared from sequencesidentified according to Example 1 using either a full contig sequence inthe case of SEQ ID Nos: 1-6, or a sequence amplified from the assembledcontig using the primer pairs as set forth in the sequence listing.Varying does of dsRNA were applied as an overlay to corn rootwormartificial diet according to the following procedure. Diabroticavirgifera virgifera (WCR) eggs were obtained from Crop Characteristics,Inc., Farmington, Minn. The non-diapausing WCR eggs were incubated insoil for about 13 days at 24C, 60% relative humidity, in completedarkness. On day 13 the soil containing WCR eggs was placed between #30and #60 mesh sieves and the eggs were washed out of the soil using ahigh pressure garden hose. The eggs were surface disinfested by soakingin LYSOL for three minutes, rinsed three times with sterile water,washed one time with a 10% formalin solution and then rinsed threeadditional times in sterile water. Eggs treated in this way weredispensed onto sterile coffee filters and hatched overnight at 27° C.,60% relative humidity, in complete darkness.

To prepare dsRNA, amplicons of selected sequences were cloned into aplasmid vector capable of replication in E. coli and sufficient amountsof plasmid DNA was recovered to allow for in vitro T7 RNA polymerasetranscription from the embedded convergent T7 promoters at either end ofthe cloned fragment. Double stranded RNA was produced and subjected tobioassay; one RNA segment comprising the sequence as set forth in thesequence listing, the other RNA segment being substantially the reversecomplement of the nucleotide sequence, uridines appropriately positionedin place of thymidines. A sample of double stranded RNA (dsRNA) wastreated with DICER or with RNAse III to produce sufficient quantities ofsmall interfering RNA's (siRNA). Samples containing siRNA or dsRNA wereoverlayed onto CRW diet bioassay as described above and larvae wereallowed to feed as set forth below.

A sample of double stranded RNA was either added directly to each wellcontaining insect diet as indicated above, or was modified prior tobeing added to insect diet. Modification of double stranded RNA followedthe instructions for RNAse III (AMBION CORPORATION, Austin, Tex.) orDICER (STRATAGENE, La Jolla, Calif.) provided by the manufacturer. RNAseIII digestion of double stranded RNA produced twenty-one and twenty-twonucleotide duplexes containing 5′ phosphorylated ends and 3′ hydroxylends with 2-3 base overhangs, similar to the ˜21-26 base pair duplexedshort interfering RNA (siRNA) fragments produced by the dicer enzyme inthe eukaryotic pathway identified by Hamilton et. al. (1999) andElbashir et. al. (2001a). This collection of short interfering RNAduplexes was further purified and a sample characterized bypolyacrylamide gel electrophoresis to determine the integrity andefficiency of duplex formation. The purity and quantity of the samplewas then determined by spectrophotometry at a wavelength of 250nanometers, and unused sample retained for further use by storage at˜20° C.

Insect diet was prepared essentially according to Pleau et al. (2002),with the following modifications. 9.4 grams of SERVA agar was dispensedinto 540 milliliters of purified water and agitated until the agar wasthoroughly distributed. The water/agar mixture was heated to boiling tocompletely dissolve the agar, and then poured into a WARING blender. Theblender was maintained at low speed while 62.7 grams of BIO-SERV DIETmix (F9757), 3.75 grams lyophilized corn root, 1.25 milliliters of greenfood coloring, and 0.6 milliliters of formalin was added to the hot agarmixture. The mixture was then adjusted to pH 9.0 with the addition of a10% potassium hydroxide stock solution. The approximately 600 millilitervolume of liquid diet was continually mixed at high speed and maintainedat from about 48° C. to about 60° C. using a sterilized NALGENE coatedmagnetic stir bar on a magnetic stirring hot plate while being dispensedin aliquots of 200 microliters into each well of FALCON 96-well roundbottom microtiter plates. The diet in the plates was allowed to solidifyand air dry in a sterile biohood for about ten minutes.

Thirty (30) microliter volumes of test samples containing either controlreagents or double stranded RNA in varying quantities was overlayed ontothe surface of the insect diet in each well using a micro-pipettorrepeater. Insect diet was allowed to stand in a sterile biohood for upto one half hour after application of test samples to allow the reagentsto diffuse into the diet and to allow the surface of the diet to dry.One WCR neonate larva was deposited to each well with a fine paintbrush.Plates were then sealed with MYLAR and ventilated using an insect pin.12-72 insect larvae were tested per dose depending on the design of theassay. The bioassay plates were incubated at 27° C., 60% relativehumidity in complete darkness for 12-14 days. The number of survivinglarvae per dose was recorded at the 12-14 day time point. Larval masswas determined using a suitable microbalance for each surviving larva.Data was analyzed using JMP®4 statistical software (SAS Institute, 1995)and a full factorial ANOVA was conducted with a Dunnet's test to lookfor treatment effects compared to the untreated control (P<0.05). ATukey-Kramer post hoc test was performed to compare all pairs of thetreatments (P<0.05). The results of the CRW larvae feeding assaysexhibited significant growth inhibition and mortality compared tocontrols as explained below.

Example 3 Results of Insect Feeding Bioassays

Artificial diet sufficient for rearing corn rootworm larvae was preparedby applying samples of double stranded RNA sequences identified asdescribed in Example 1 using bioassays carried out as described inExample 2. Corn rootworm larvae were typically allowed to feed on thediet for twelve days and mortality and stunting monitored in comparisonto rootworms allowed to feed only on negative and positive controldiets. The results of the studies confirmed significant levels (p<0.05)of larval stunting and/or mortality using dsRNAs containing portionssequences homologous to a variety of different gene classes. Thesequences and vectors yielding significant stunting and/or mortality andthe corresponding SEQ ID NO for the sequence expressed as a dsRNA aregiven in Tables 1-5 below.

TABLE 1 dsRNA Constructs Demonstrating Significant Stunting and/orMortality in Effect in Insect Feeding Bioassays with Southern CornRootworm or Western Corn Rootworm Vector Sequence Expressed as dsRNA SEQID NO RNAi-pIC17553:001 Apple 697 RNAi-pIC17504:049 EST Full LengthV-ATPase 695 RNAi-pIC17554:001 Rpl 9 698 RNAi-pIC17555:001 Rpl 19 699RNAi-pIC17504:050 Section 6.0 V-ATPase 711 RNAi-pIC19514:001 WCR mRNAcapping enzyme LIB5496-028-A1-M1-A7 696 RNAi-pIC17552:003LIB5462-042-A1-M1-H10 700 Dv.6_CG9355 DUSKY STRUCTURAL CONSTITUENT OFCUTICLE RNAi-pIC17546:003 LIB5462-091-A1-M1-G3 701 Dv.1_CG6217 KNICKKOPFUNK RNAi-pIC17546:001 Dv.1_CG6217 1 RNAi-pIC17549:001 Dv.4_CG1435 4RNAi-pIC17550:001 LIB5444-065-A1-M1-D5 5 Dv.5_cg1915_1 RNAi-pIC17551:001LIB5462-012-A1-M2-B2 703 Dv.5_cg1915_2 RNAi-pIC17552:001 Dv.6_CG9355 6RNAi-pMON78412:002 Dv.7_CG3416; putative Mov34:CG3416 10 orthologRNAi-pMON96172:002 Dv.8_CG1088; putative vacuolar 14 H+ATPase E subunit:CG1088 ortholog RNAi-pMON96168:002 Dv.9_CG2331; putative ATPaseactivity: 18 CG2331 ortholog RNAi-pMON78424:002 Dv.10_CG6141; putativeribosomal protein 22 L9: CG6141 ortholog RNAi-pMON78425:001 Dv.11_CG274626 RNAi-pMON78444:002 Dv.12_CG1341; putative proteasome 30 regulatoryparticle, rpt1: CG1341 ortholog RNAi-pMON78416:002 Dv.13_CG11276;putative ribosomal 34 protein S4: CG11276 ortholog RNAi-pMON78434:001Dv.14_CG17927_2; putative myosin heavy 38 chain: CG17927 orthologRNAi-pMON78439:001 Dv.16_CG5394; putative glutamyl-prolyl- 46 tRNAsynthetase: CG5394 ortholog RNAi-pMON78438:001 Dv.17_CG10149; putativeproteasome 50 p44.5 subunit, rpn6: CG10149 ortholog RNAi-pMON78435:002Dv.18_CG1404; putative RAN small 54 monomeric GTPase: CG1404 orthologRNAi-pMON78449:002 Dv.19_CG18174; putative proteasome 58 regulatoryparticle, lid subcomplex, rpn11: CG18174 ortholog RNAi-pMON78419:001Dv.20_CG3180_1; putative DNA-directed 62 RNA polymerase II: CG3180ortholog RNAi-pMON78440:002 Dv20_CG3180_2; putative DNA-directed 706 RNApolymerase II: CG3180 ortholog RNAi-pMON78420:001 Dv.21_CG3320; putativeRab1: CG3320 70 ortholog RNAi-pMON78410:002 Dv.22_CG3395; putativeRibosomal 74 protein S9: CG3395 ortholog RNAi-pMON78422:001Dv.23_CG7269; putative helicase: 78 CG7269 ortholog RNAi-pMON78423:001Dv.25_CG9012; putative Clathrin heavy 86 chain: CG9012 orthologRNAi-pMON78414:006 DV.26 sec1 ~5′ half of EST 710 RNAi-pMON78414:001Dv.26_CG9261; putative 90 sodium/potassium-exchanging ATPase: CG9261ortholog RNAi-pMON78413:001 Dv.27_CG12052; putative RNA 94 polymerase IItranscription factor: CG12052 ortholog. RNAi-pMON78427:001 Dv.35_CG3762;putative Vha68-2: 126 CG3762 ortholog. RNAi-pMON97122:001 C1_Dv.35;putative Vha68-2: CG3762 713 ortholog; concatamer RNAi-pMON97127:001C2_Dv.35; putative Vha68-2: CG3762 714 ortholog; concatamerRNAi-pMON97125:001 C3_Dv.35; putative Vha68-2: CG3762 715 ortholog;concatamer RNAi-pMON78441:001 Dv.39_CG9078; putative sphingolipid 142delta-4 desaturase; stearoyl-CoA 9- desaturase: CG9078 orthologRNAi-pMON97114:001 Dv.41_CG2637; Female sterile Ketel; 150 involved inprotein-nucleus import: CG2637 ortholog RNAi-pMON97140:001 Dv.44_CG1244;Putative nucleic acid 162 binding activity: CG1244 orthologRNAi-pMON78429:001 Dv.46_CG10689; putative RNA helicase: 170 CG10689ortholog. RNAi-pMON78432:001 Dv.48_CG33196; putative transmembrane 178receptor protein tyrosine kinase: CG33196 ortholog RNAi-pMON78428:001Dv.49_CG8055_1; putative binding, 182 carrier activity: CG8055_1ortholog RNAi-pMON78428:003 Dv.49_CG8055_1; putative binding, 704carrier activity: CG8055_1 ortholog Free region selected from DV.49RNAi-pMON78426:001 Dv.50_CG10110_1; putative Cleavage and 186polyadenylation specificity factor: CG10110_1 orthologRNAi-pMON96185:001 Dv.55_CG5931; putative splicing factor 202 activity,RNA helicase activity: CG5931 ortholog RNAi-pMON78442:001 Dv.57_CG2968putative hydrogen- 206 exporting ATPase: CG2968 orthologRNAi-pMON78431:001 Dv.58_CG1751; putative signal peptidase: 210 CG1751ortholog RNAi-pMON96177:001 Dv.61_CG3725_1; putative Calcium 222 ATPase:CG3725 ortholog RNAi-pMON96183:001 Dv.62_CG3612; putative bellwether:230 CG3612 ortholog RNAi-pMON96180:002 Dv.65_CG7033; putative chaperone242 activity: CG7033 ortholog RNAi-pMON96176:001 Dv.66_CG32019; putativebent: CG32019 246 ortholog RNAi-pMON96170:002 Dv.67_CG16916; putativeendopeptidase 250 activity: CG16916 ortholog RNAi-pMON96166:001Dv.70_CG5771; putative Rab-protein 11: 258 CG5771 orthologRNAi-pMON96179:001 Dv.72_CG6831; putative rhea: CG6831 266 orthologRNAi-pMON96186:001 Dv.73_CG10119; putative Lamin C: 270 CG10119 orthologRNAi-pMON96160:002 Dv.74_CG6375; putative pitchoune: 274 CG6375 orthologRNAi-pMON97137:001 Dv.77_CG4214; putative Syntaxin 5; 286 involved inintracellular protein transport: CG4214 ortholog RNAi-pMON96167:001Dv.82_CG8264; putative Bx42: CG8264 302 ortholog RNAi-pMON96171:001Dv.83_CG11397; putative gluon: 306 CG11397 ortholog RNAi-pMON96187:003Dv.85_CG4494; putative protein binding 314 activity: CG4494 orthologRNAi-pMON96174:001 Dv.86_CG5055; putative bazooka: 318 CG5055 orthologRNAi-pMON97126:001 Dv.88_CG8756; function unknown; 326 contains chitinbinding domain: CG8756 ortholog RNAi-pMON97130:001 Dv.93_CG8515;putative structural 342 constituent of cuticle; contains chitin bindingdomain: CG8515 ortholog RNAi-pMON97109:001 Dv.99_CG2446; Unknown; lethalin 366 Drosophila & low homology with human: CG2446 orthologRNAi-pMON97111:001 Dv.105_CG1250_1; GTPase activator, 390 involved inintracellular protein transport: CG1250 ortholog RNAi-pMON97112:001Dv.105_CG1250_2; GTPase activator, 394 involved in intracellular proteintransport: CG1250 ortholog RNAi-pMON97107:001 Dv.107_CG14813; COPIvesicle coat; 398 involved in Golgi to ER intracellular proteintransport: CG14813 ortholog RNAi-pMON97115:001 Dv.108_CG17248;n-synaptobrevin; 402 involved in intracellular protein transport:CG17248 ortholog RNAi-pMON97133:001 Dv.113; function unknown; WCR unique422 sequence RNAi-pMON97121:001 Dv.122_CG3164; putative ATP-binding 454cassette transporter activity: CG3164 ortholog RNAi-pMON97134:001Dv.127; function unknown; no homology 470 with human RNAi-pMON97171:001Dv.146; function unknown, WCR unique 514 sequence RNAi-pMON97166:001Dv.147; function unknown, WCR unique 518 sequence RNAi-pMON97167:001Dv.149; function unknown, WCR unique 526 sequence RNAi-pMON97169:001Dv.155; function unknown, WCR unique 550 sequence RNAi-pMON97173:001Dv.162; function unknown, WCR unique 578 sequence RNAi-pMON97170:001Dv.170; function unknown, WCR unique 610 sequence

TABLE 2 dsRNA Constructs Causing Significant Stunting Levels in FeedingBioassays with Western Corn Rootworm (WCR) Larvae Vector SequenceExpressed as dsRNA RNAi-pIC17553:001 Apple RNAi-pIC17504:049 EST FullLength V-ATPase RNAi-pIC17554:001 Rpl 9 RNAi-pIC17555:001 Rpl 19RNAi-pIC17504:050 Section 6.0 V-ATPase RNAi-pIC16005:001 V-ATPase Dsubunit 1 RNAi-pIC19514:001 WCR mRNA capping enzyme LIB5496-028-A1-M1-A7RNAi-pIC17546:001 Dv.1_CG6217 RNAi-pIC17549:001 Dv.4_CG1435RNAi-pIC17551:001 LIB5462-012-A1-M2-B2 Dv.5_cg1915_2 RNAi-pIC17552:001Dv.6_CG9355 RNAi-pMON78412:001 Dv.7_CG3416 RNAi-pMON78412:002Dv.7_CG3416; putative Mov34:CG3416 ortholog RNAi-pMON96172:002Dv.8_CG1088; putative vacuolar H+ATPase E subunit: CG1088 orthologRNAi-pMON96168:002 Dv.9_CG2331; putative ATPase activity: CG2331ortholog RNAi-pMON78424:002 Dv.10_CG6141; putative ribosomal protein L9:CG6141 ortholog RNAi-pMON78425:001 Dv.11_CG2746 RNAi-pMON78444:002Dv.12_CG1341; putative proteasome regulatory particle, rpt1: CG1341ortholog RNAi-pMON78416:002 Dv.13_CG11276; putative ribosomal proteinS4: CG11276 ortholog RNAi-pMON78434:001 Dv.14_CG17927_2; putative myosinheavy chain: CG17927 ortholog RNAi-pMON78439:001 Dv.16_CG5394; putativeglutamyl-prolyl-tRNA synthetase: CG5394 ortholog RNAi-pMON78435:002Dv.18_CG1404; putative RAN small monomeric GTPase: CG1404 orthologRNAi-pMON78449:002 Dv.19_CG18174; putative proteasome regulatoryparticle, lid subcomplex, rpn11: CG18174 ortholog RNAi-pMON78440:002Dv.20_CG3180_2; putative DNA-directed RNA polymerase II: CG3180 orthologRNAi-pMON78420:002 Dv.21_CG3320; putative Rab1: CG3320 orthologRNAi-pMON78410:001 Dv.22_CG3395; putative Ribosomal protein S9: CG3395ortholog RNAi-pMON78422:001 Dv.23_CG7269; putative helicase: CG7269ortholog RNAi-pMON78423:001 Dv.25_CG9012; putative Clathrin heavy chain:CG9012 ortholog RNAi-pMON78414:001 Dv.26_CG9261; putativesodium/potassium-exchanging ATPase: CG9261 ortholog RNAi-pMON78413:001Dv.27_CG12052; putative RNA polymerase II transcription factor: CG12052ortholog. RNAi-pMON97122:001 C1_Dv.35; putative Vha68-2: CG3762ortholog; concatamer RNAi-pMON97127:001 C2_Dv.35; putative Vha68-2:CG3762 ortholog; concatamer RNAi-pMON97125:001 C3_Dv.35; putativeVha68-2: CG3762 ortholog; concatamer RNAi-pMON78427:007 Dv.35_CG3762;putative Vha68-2: CG3762 ortholog. RNAi-pMON78441:001 Dv.39_CG9078;putative sphingolipid delta-4 desaturase; stearoyl- CoA 9-desaturase:CG9078 ortholog RNAi-pMON97114:001 Dv.41_CG2637; Female sterile Ketel;involved in protein-nucleus import: CG2637 ortholog RNAi-pMON78429:001Dv.46_CG10689; putative RNA helicase: CG10689 ortholog.RNAi-pMON78432:001 Dv.48_CG33196; putative transmembrane receptorprotein tyrosine kinase: CG33196 ortholog RNAi-pMON78428:001Dv.49_CG8055_1; putative binding, carrier activity: CG8055_1 orthologRNAi-pMON78428:003 Dv.49_CG8055_1; putative binding, carrier activity:CG8055_1 ortholog Free region selected from DV.49 RNAi-pMON78426:001Dv.50_CG10110_1; putative Cleavage and polyadenylation specificityfactor: CG10110_1 ortholog RNAi-pMON96185:001 Dv.55_CG5931; putativesplicing factor activity, RNA helicase activity: CG5931 orthologRNAi-pMON78442:001 Dv.57_CG2968; putative hydrogen-exporting ATPase:CG2968 ortholog RNAi-pMON78431:001 Dv.58_CG1751; putative signalpeptidase: CG1751 ortholog RNAi-pMON96177:001 Dv.61_CG3725_1; putativeCalcium ATPase: CG3725 ortholog RNAi-pMON96182:001 Dv.61_CG3725_2;putative Calcium ATPase: CG3725 ortholog RNAi-pMON96183:001Dv.62_CG3612; putative bellwether: CG3612 ortholog RNAi-pMON96180:002Dv.65_CG7033; putative chaperone activity: CG7033 orthologRNAi-pMON96180:001 Dv.65_CG7033; putative chaperone activity: CG7033ortholog RNAi-pMON96176:001 Dv.66_CG32019; putative bent: CG32019ortholog RNAi-pMON96170:002 Dv.67_CG16916; putative endopeptidaseactivity: CG16916 ortholog RNAi-pMON96166:001 Dv.70_CG5771; putativeRab-protein 11: CG5771 ortholog RNAi-pMON96179:001 Dv.72_CG6831;putative rhea: CG6831 ortholog RNAi-pMON96186:001 Dv.73_CG10119;putative Lamin C: CG10119 ortholog RNAi-pMON96160:002 Dv.74_CG6375;putative pitchoune: CG6375 ortholog RNAi-pMON96160:001 Dv.74_CG6375;putative pitchoune: CG6375 ortholog RNAi-pMON97137:002 Dv.77_CG4214;putative Syntaxin 5; involved in intracellular protein transport: CG4214ortholog RNAi-pMON96167:001 Dv.82_CG8264; putative Bx42: CG8264 orthologRNAi-pMON96171:001 Dv.83_CG11397; putative gluon: CG11397 orthologRNAi-pMON96187:003 Dv.85_CG4494; putative protein binding activity:CG4494 ortholog RNAi-pMON97126:001 Dv.88_CG8756; function unknown;contains chitin binding domain: CG8756 ortholog RNAi-pMON97109:001Dv.99_CG2446; Unknown; lethal in Drosophila & low homology with human:CG2446 ortholog RNAi-pMON97111:001 Dv.105_CG1250_1; GTPase activator,involved in intracellular protein transport: CG1250 orthologRNAi-pMON97107:001 Dv.107_CG14813; COPI vesicle coat; involved in Golgito ER intracellular protein transport: CG14813 orthologRNAi-pMON97115:001 Dv.108_CG17248; n-synaptobrevin; involved inintracellular protein transport: CG17248 ortholog RNAi-pMON97121:001Dv.122_CG3164; putative ATP-binding cassette transporter activity:CG3164 ortholog RNAi-pMON97171:001 Dv.146; function unknown, WCR uniquesequence RNAi-pMON97166:001 Dv.147; function unknown, WCR uniquesequence RNAi-pMON97167:001 Dv.149; function unknown, WCR uniquesequence RNAi-pMON97169:001 Dv.155; function unknown, WCR uniquesequence RNAi-pMON97173:001 Dv.162; function unknown, WCR uniquesequence RNAi-pMON97170:001 Dv.170; function unknown, WCR uniquesequence

TABLE 3 dsRNA Constructs Causing Significant Mortality Levels in FeedingBioassays with Western Corn Rootworm (WCR) Larvae Vector SequenceExpressed as dsRNA RNAi-pIC17553:001 Apple RNAi-pIC17504:049 EST FullLength V-ATPase RNAi-pIC17555:001 Rpl 19 RNAi-pIC17554:001 Rpl 9RNAi-pIC17504:050 Section 6.0 V-ATPase RNAi-pIC17504:054 V-ATPasesubunit 2 sequence from Diabrotica virgifera virgifera Full EST sequenceserving as positive control RNAi-pIC19514:001 WCR mRNA capping enzymeLIB5496-028-A1-M1-A7 RNAi-pIC17546:001 Dv.1_CG6217 RNAi-pIC17549:001Dv.4_CG1435 RNAi-pIC17550:001 LIB5444-065-A1-M1-D5 Dv.5_cg1915_1RNAi-pMON78412:002 Dv.7_CG3416; putative Mov34:CG3416 orthologRNAi-pMON96172:001 Dv.8_CG1088; putative vacuolar H+ATPase E subunit:CG1088 ortholog RNAi-pMON96168:001 Dv.9_CG2331; putative ATPaseactivity: CG2331 ortholog RNAi-pMON78424:002 Dv.10_CG6141; putativeribosomal protein L9: CG6141 ortholog RNAi-pMON78425:001 Dv.11_CG2746RNAi-pMON78444:001 Dv.12_CG1341; putative proteasome regulatoryparticle, rpt1: CG1341 ortholog RNAi-pMON78416:002 Dv.13_CG11276;putative ribosomal protein S4: CG11276 ortholog RNAi-pMON78434:001Dv.14_CG17927_2; putative myosin heavy chain: CG17927 orthologRNAi-pMON78435:001 Dv.18_CG1404; putative RAN small monomeric GTPase:CG1404 ortholog RNAi-pMON78449:001 Dv.19_CG18174; putative proteasomeregulatory particle, lid subcomplex, rpn11: CG18174 orthologRNAi-pMON78440:001 Dv.20_CG3180; putative DNA-directed RNA polymeraseII: CG3180 ortholog RNAi-pMON78420:001 Dv.21_CG3320; putative Rab1:CG3320 ortholog RNAi-pMON78410:001 Dv.22_CG3395; putative Ribosomalprotein S9: CG3395 ortholog RNAi-pMON78422:001 Dv.23_CG7269; putativehelicase: CG7269 ortholog RNAi-pMON78414:006 Dv.26 sec1 ~5′ half of ESTRNAi-pMON78414:001 Dv.26_CG9261; putative sodium/potassium-exchangingATPase: CG9261 ortholog RNAi-pMON97122:001 C1_Dv.35; putative Vha68-2:CG3762 ortholog; concatamer RNAi-pMON97127:001 C2_Dv.35; putativeVha68-2: CG3762 ortholog; concatamer RNAi-pMON97125:001 C3_Dv.35;putative Vha68-2: CG3762 ortholog; concatamer RNAi-pMON78427:001Dv.35_CG3762; putative Vha68-2: CG3762 ortholog. RNAi-pMON97114:001Dv.41_CG2637; Female sterile Ketel; involved in protein-nucleus import:CG2637 ortholog RNAi-pMON97140:001 Dv.44_CG1244; Putative nucleic acidbinding activity: CG1244 ortholog RNAi-pMON78429:001 Dv.46_CG10689;putative RNA helicase: CG10689 ortholog. RNAi-pMON78428:001Dv.49_CG8055_1; putative binding, carrier activity: CG8055_1 orthologRNAi-pMON78428:003 Dv.49_CG8055_1; putative binding, carrier activity:CG8055_1 ortholog Free region selected from DV.49 RNAi-pMON78426:001Dv.50_CG10110_1; putative Cleavage and polyadenylation specificityfactor: CG10110_1 ortholog RNAi-pMON96185:001 Dv.55_CG5931; putativesplicing factor activity, RNA helicase activity: CG5931 orthologRNAi-pMON78431:001 Dv.58_CG1751; putative signal peptidase: CG1751ortholog RNAi-pMON96177:001 Dv.61_CG3725_1; putative Calcium ATPase:CG3725 ortholog RNAi-pMON96182:001 Dv.61_CG3725_2; putative CalciumATPase: CG3725 ortholog RNAi-pMON96180:001 Dv.65_CG7033; putativechaperone activity: CG7033 ortholog RNAi-pMON96176:001 Dv.66_CG32019;putative bent: CG32019 ortholog RNAi-pMON96170:001 Dv.67_CG16916;putative endopeptidase activity: CG16916 ortholog RNAi-pMON96166:001Dv.70_CG5771; putative Rab-protein 11: CG5771 orthologRNAi-pMON96160:001 Dv.74_CG6375; putative pitchoune: CG6375 orthologRNAi-pMON97137:001 Dv.77_CG4214; putative Syntaxin 5; involved inintracellular protein transport: CG4214 ortholog RNAi-pMON96167:001Dv.82_CG8264; putative Bx42: CG8264 ortholog RNAi-pMON96187:002Dv.85_CG4494; putative protein binding activity: CG4494 orthologRNAi-pMON97126:001 Dv.88_CG8756; function unknown; contains chitinbinding domain: CG8756 ortholog RNAi-pMON97130:001 Dv.93_CG8515;putative structural constituent of cuticle; contains chitin bindingdomain: CG8515 ortholog RNAi-pMON97111:001 Dv.105_CG1250_1; GTPaseactivator, involved in intracellular protein transport: CG1250 orthologRNAi-pMON97107:001 Dv.107_CG14813; COPI vesicle coat; involved in Golgito ER intracellular protein transport: CG14813 orthologRNAi-pMON97115:001 Dv.108_CG17248; n-synaptobrevin; involved inintracellular protein transport: CG17248 ortholog RNAi-pMON97133:001Dv.113; function unknown; WCR unique sequence RNAi-pMON97121:001Dv.122_CG3164; putative ATP-binding cassette transporter activity:CG3164 ortholog RNAi-pMON97134:001 Dv.127; function unknown; no homologywith human

TABLE 4 dsRNA Constructs Causing Significant Stunting Levels in FeedingBioassays with Southern Corn Rootworm (SCR) Larvae Vector SequenceExpressed as dsRNA RNAi-pMON96172:001 Dv8_CG1088; putative vacuolarH+ATPase E subunit: CG1088 ortholog RNAi-pMON96168:001 Dv9_CG2331;putative ATPase activity: CG2331 ortholog RNAi-pMON78424:001Dv.10_CG6141 RNAi-pMON96155:002 Dv.10_CG6141; putative ribosomal proteinL9: CG6141 ortholog. RNAi-pMON78425:001 Dv.11_CG2746 RNAi-pMON96158:002Dv.11_CG2746; putative Ribosomal protein L19: CG2746 ortholog.RNAi-pMON78416:001 Dv.13_CG11276 RNAi-pMON78434:001 Dv.14_CG17927_2;putative myosin heavy chain: CG17927 ortholog RNAi-pMON78435:001Dv.18_CG1404; putative RAN small monomeric GTPase: CG1404 orthologRNAi-pMON78449:001 Dv.19_CG18174; putative proteasome regulatoryparticle, lid subcomplex, rpn11: CG18174 ortholog RNAi-pMON78419:001Dv.20_CG3180 RNAi-pMON78420:001 Dv.21_CG3320; putative Rab1: CG3320ortholog RNAi-pMON78414:001 Dv.26_CG9261; putativesodium/potassium-exchanging ATPase: CG9261 ortholog RNAi-pMON97122:001C1_Dv35; putative Vha68-2: CG3762 ortholog; concatamerRNAi-pMON97125:001 C3_Dv35; putative Vha68-2: CG3762 ortholog;concatamer RNAi-pMON78427:008 Dv.35_CG3762; putative Vha68-2: CG3762ortholog. RNAi-pMON78428:001 Dv.49_CG8055_1; putative binding, carrieractivity: CG8055_1 ortholog RNAi-pMON78442:001 Dv.57_CG2968; putativehydrogen-exporting ATPase: CG2968 ortholog RNAi-pMON96177:001Dv61_CG3725_1; putative Calcium ATPase: CG3725 orthologRNAi-pMON96166:001 Dv70_CG5771; putative Rab-protein 11: CG5771 orthologRNAi-pMON97126:001 Dv88_CG8756; function unknown; contains chitinbinding domain: CG8756 ortholog RNAi-pMON97111:001 Dv105_CG1250_1;GTPase activator, involved in intracellular protein transport: CG1250ortholog RNAi-pMON97112:001 Dv105_CG1250_2; GTPase activator, involvedin intracellular protein transport: CG1250 ortholog RNAi-pMON97107:001Dv107_CG14813; COPI vesicle coat; involved in Golgi to ER intracellularprotein transport: CG14813 ortholog RNAi-pMON97121:001 Dv122_CG3164;putative ATP-binding cassette transporter activity: CG3164 ortholog

TABLE 5 dsRNA Constructs Causing Significant Mortality Levels in FeedingBioassays with Southern Corn Rootworm (SCR) Larvae Vector SequenceExpressed as dsRNA RNAi-pIC17546:003 LIB5462-091-A1-M1-G3 Dv1_CG6217KNICKKOPF UNK RNAi-pIC17504:055 V-ATPase subunit 2 sequence fromDiabrotica virgifera virgifera Full EST sequence serving as positivecontrol RNAi-pMON96155:001 Dv.10_CG6141; putative ribosomal protein L9:CG6141 ortholog RNAi-pMON96158:001 Dv.11_CG2746; putative Ribosomalprotein L19: CG2746 ortholog RNAi-pMON78416:001 Dv.13_CG11276RNAi-pMON96154:003 Dv.14_CG17927; putative myosin heavy chain: CG17927ortholog; cells grown in S Complete medium. RNAi-pMON78438:001Dv.17_CG10149; putative proteasome p44.5 subunit, rpn6: CG10149 orthologRNAi-pMON78449:001 Dv.19_CG18174; putative proteasome regulatoryparticle, lid subcomplex, rpn11: CG18174 ortholog RNAi-pMON78440:001Dv.20_CG3180; putative DNA-directed RNA polymerase II: CG3180 orthologRNAi-pMON96156:001 Dv.20_CG3180; putative RNA polymerase II 140 kDsubunit: CG3180 ortholog RNAi-pMON78420:001 Dv.21_CG3320; putative Rab1:CG3320 ortholog RNAi-pMON78427:006 Dv.35_CG3762; putative Vha68-2:CG3762 ortholog. RNAi-pMON78428:001 Dv.49_CG8055_1; putative binding,carrier activity: CG8055_1 ortholog RNAi-pMON96177:001 Dv61_CG3725_1;putative Calcium ATPase: CG3725 ortholog RNAi-pMON96166:001 Dv70_CG5771;putative Rab-protein 11: CG5771 ortholog RNAi-pMON96174:001 Dv86_CG5055;putative bazooka: CG5055 ortholog RNAi-pMON97111:001 Dv105_CG1250_1;GTPase activator, involved in intracellular protein transport: CG1250ortholog RNAi-pMON97107:001 Dv107_CG14813; COPI vesicle coat; involvedin Golgi to ER intracellular protein transport: CG14813 ortholog

Example 4 Transgenic Plant Transformation and Bioassays

Briefly, the sequence encoding a dsRNA construct as described above islinked at the 5′ end to a sequence consisting of a 35S promoter operablylinked to a maize hsp70 intron and at the 3′ end to a Nos3′transcription termination and polyadenylation sequence. This expressioncassette is placed downstream of a glyphosate selection cassette. Theselinked cassettes are then placed into an Agrobacterium tumefaciens planttransformation functional vector, used to transform maize tissue toglyphosate tolerance, and events selected and transferred to soil. R₀plant roots are fed to western corn rootworm larvae (WCR, Diabroticavirgifera). Transgenic corn roots are handed-off in Petri dishes withMS0D medium containing antibiotics and glyphosate for in vitroselection. Two WCR larvae are infested per root in each dish with a finetip paintbrush. The dishes are sealed with Parafilm to prevent thelarvae from escaping. The assays are placed into a 27° C., 60% RHPercival incubator in complete darkness. Contamination and larvalquality are monitored. After six days of feeding on root tissue, thelarvae are transferred to WCR diet in a 96 well plate. The larvae areallowed to feed on the diet for eight days making the full assayfourteen days long. Larval mass and survivorship are recorded foranalysis. A one-way ANOVA analysis and a Dunnett's test is performed onthe larval mass data to look for statistical significance compared to anuntransformed negative control. WCR larvae stunting is measured afterfeeding on two events and compared to growth of larvae fed on negativecontrol plants.

Transgenic corn plants (R₀) generated are planted into 10-inch potscontaining Metromix soil after reaching an appropriate size. When plantsreach the V4 growth stage, approximately 1000 Western corn rootworm(WCR, Diabrotica virgifera) eggs are infested into the root zone.Non-transgenic corn of the same genotype is infested at a similar growthstage to serve as a negative control. Eggs are pre-incubated so hatchoccurs within 24 hours of infestation. Larvae are allowed to feed on theroot systems for 3 weeks. Plants are removed from the soil and washed sothat the roots can be evaluated for larval feeding. Root damage is ratedusing a Node Injury Scale (NIS) to score the level of damage where a 0indicates no damage, a 1 indicates that one node of roots is pruned towithin 1.5 inches, a 2 indicates that 2 nodes are pruned, while a 3indicates that 3 nodes are pruned. Because the plants being used forevaluation are directly out of tissue culture after transformation andbecause transformation events are unique, only a single plant isevaluated per event at this time. The plants in the assay that presentsigns or symptoms of larval feeding indicate that a successfulinfestation is obtained. Negative control plant roots are moderately toseverely damaged averaging whereas roots of the transgenic plantsprovide substantial control of larval feeding, with about 0.2 or less onthe Node Injury Scale.

Example 5 Implementing Insect Pest Gene Suppression Using A ta-siRNAMediated Silencing Method

An alternative method to silence genes in a plant pest uses the recentlydiscovered class of trans-acting small interfering RNA (ta-siRNA)(Dalmay et al., 2000; Mourrain et al., 2000; Peragine et al, 2004;Vazquez et al, 2004). ta-siRNA are derived from single strand RNAtranscripts that are targeted by naturally occurring miRNA within thecell. Methods for using microRNA to trigger ta-siRNA for gene silencingin plants are described in U.S. Provisional Patent Application Ser. No.60/643,136 (Carrington et al. 2004), incorporated herein by reference inits entirety. At least one pest specific miRNA expressed in gutepithelial cells of corn rootworm larvae is identified. This pestspecific miRNA is then used to identify at least one target RNAtranscript sequence complementary to the miRNA that is expressed in thecell. The corresponding target sequence is a short sequence of no morethan 21 contiguous nucleotides that, when part of a RNA transcript andcontacted by its corresponding miRNA in a cell type with a functionalRNAi pathway, leads to slicer-mediated cleavage of said transcript. OncemiRNA target sequences are identified, at least one miRNA targetsequence is fused to a second sequence that corresponds to part of apest gene that is to be silenced using this method. For example, themiRNA target sequence(s) is fused to any of SEQ ID NO:1 through SEQ IDNO:906, or a fragment thereof, such as a sequence of the corn rootwormvacuolar ATPase (V-ATPase) gene. The miRNA target sequence can be placedat the 5′ end, the 3′ end, or embedded in the middle of the targetsequence. It may be preferable to use multiple miRNA target sequencescorresponding to multiple miRNA genes, or use the same miRNA targetsequence multiple times in the chimera of the miRNA target sequence andthe target gene sequence. The target gene sequence can be of any length,with a minimum of 21 bp.

The chimera of the miRNA target sequence(s) and the target gene sequenceis expressed in plant cells using any of a number of appropriatepromoter and other transcription regulatory elements, as long as thetranscription occurs in cell types subject to being provided in the dietof the pest, e.g. corn roots for control of corn rootworm.

This method may have the additional advantage of delivering longer RNAmolecules to the target pest. Typically, dsRNAs produced in plants arerapidly processed by Dicer into short RNA's that may not be effectivewhen fed exogenously to some pests. In this method, a single strandtranscript is produced in the plant cell, taken up by the pest, andconverted into a dsRNA in the pest cell where it is then processed intota-siRNA capable of post-transcriptionally silencing one or more genesin one or more target pests.

Example 6 Method for Providing a DNA Sequence for dsRNA-Mediated GeneSilencing

This example illustrates a method for providing a DNA sequence fordsRNA-mediated gene silencing. More specifically, this example describesselection of an improved DNA useful in dsRNA-mediated gene silencing by(a) selecting from a target gene an initial DNA sequence including morethan 21 contiguous nucleotides; (b) identifying at least one shorter DNAsequence derived from regions of the initial DNA sequence consisting ofregions predicted to not generate undesirable polypeptides and notexhibiting identity with known sequences such as homologs/orthologs, and(c) selecting a DNA sequence for dsRNA-mediated gene silencing thatincludes the at least one shorter DNA sequence. Undesirable polypeptidesinclude, but are not limited to, polypeptides homologous to allergenicpolypeptides and polypeptides homologous to known polypeptide toxins.

WCR V-ATPase has been demonstrated to function in corn rootworm feedingassays to test dsRNA mediated silencing as a means of controlling larvalgrowth. A cDNA sequence from a target gene, such as vacuolar ATPase gene(V-ATPase) from Western corn rootworm (WCR) (Diabrotica virgiferavirgifera LeConte), is selected for use as an initial DNA sequence. Thisinitial DNA sequence can be screened to identify regions within whichevery contiguous fragment including at least 21 nucleotides matchesfewer than 21 out of 21 contiguous nucleotides of known vertebratesequences. Sequence segments that are greater than about 100 contiguousnucleotides free of such 21/21 hits are identified. Thus criteriaincluding segment length, GC content, sequence, predicted function basedon sequence or function of a corresponding gene in a model organism, andpredicted secondary structure (e.g. Elbashir, et al., 2001b) may be usedto select and design sequence(s) for use. Different combinations ofthese sequence segments are combined to construct chimeric DNA sequencesfor expression as dsRNA and use in insect feeding bioassays as describedabove.

Example 7 Additional Results of Insect Feeding Bioassays with SequencesSelected from EST Database

This example illustrates additional sequences found to be effective incausing larval stunting and/or mortality when ingested by rootwormlarvae as double stranded RNA sequences. Methods for rearing cornrootworm larvae, application of dsRNA, and insect bioassays are asdescribed in Examples 1-3. The results of the studies confirmedsignificant levels (p<0.05) of larval stunting and/or mortality usingdsRNAs containing portions of sequences homologous to a variety ofdifferent gene classes. The sequences and vectors yielding significantstunting and/or mortality and the corresponding SEQ ID NO for thesequence expressed as a dsRNA are given in Table 6 below. pMON98503, anexemplary binary vector used in corn transformation, contains thefollowing elements between the right and left T-DNA borders for transferinto a plant cell: e35S-HSP70-DV49 (antisense orientation)-universalspacer-DV49 (sense orientation)-hsp17; ACT (promoter and intron)-CTP2transit signal-CP4-NOS.: pMON98504, another exemplary binary vector usedin corn transformation, contains the following elements between theright and left borders: e35S-HSP70-C1 (antisense orientation)-universalspacer-C1 (sense orientation)-hsp17; ACT (promoter and intron)-CTP2transit signal-CP4-NOS.

TABLE 6 Additional dsRNA Constructs Demonstrating Significant Stuntingand/or Mortality Effect in Insect Feeding Bioassays with Southern CornRootworm or Western Corn Rootworm SEQ ID Vector Sequence Expressed asdsRNA NO pMON98356 Dv164; function unknown, WCR unique sequence 726pMON98354 Dv172; function unknown, WCR unique sequence 727 pMON97191Dv189; function unknown, WCR unique sequence 728 pMON98359 Dv200;function unknown, WCR unique sequence 729 pMON38880 Dv207_F39H11.5;putative pbs-7, endopeptidase: F39H11.5 ortholog 730 pMON101054Dv208_F58F12_1; putative Mitochondrial F1F0-ATP synthase, subunit 731delta/ATP16: F58F12_1 ortholog pMON98437 Dv210_K11H12_2; putativerpl-15, structural constituent of ribosome: 732 K11H12_2 orthologpMON98435 Dv211_R12E2_3; putative rpn-8, translation initiation factor,26S 733 proteasome regulatory complex, subunit RPN8: R12E2_3 orthologpMON98447 Dv212_C17H12_14; putative vha-8, hydrogen-exporting ATPase:734 C17H12_14 ortholog pMON98448 Dv213_B0464_1; putative drs-1, tRNAligase: B0464_1 ortholog 735 pMON101059 Dv214_F53G12_10; putative rpl-7,structural constituent of ribosome: 736 F53G12_10 ortholog pMON98442Dv216_C52E4_4; putative rpt-1, ATPase subunit of the 19S regulatory 737complex of the proteasome: C52E4_4 ortholog pMON98441 Dv218_K01G5_4;putative ran-1, small monomeric GTPase: K01G5_4 738 ortholog pMON98440Dv219_C15H11_7; putative pas-1, endopeptidase: C15H11_7 ortholog 739pMON101081 Dv223_R10E11.1; putative cbp-1, homolog of transcriptionalcofactors 740 CBP and p300: R10E11.1 ortholog pMON101050 Dv224_F11C3_3;putative unc-54, ATP binding; motor activity: 741 F11C3_3 orthologpMON101051 Dv225_C37H5_8; putative hsp-6, heat shock protein 6: C37H5_8742 ortholog pMON38888 Dv226_C47E12.5; putative uba-1, ubiquitinactivating enzyme: 743 C47E12.5 ortholog pMON38887 Dv227_F54A3.3;putative Chaperonin complex component, TCP-1 744 gamma subunit: F54A3.3ortholog pMON101110 Dv229_D1081.8; putative Myb-like DNA binding:D1081.8 ortholog 745 pMON101052 Dv230_F55A11_2; putative syn-3, proteintransporter; syntaxin: 746 F55A11_2 ortholog pMON101107 Dv231_C30C11.1;putative mitochondrial ribosomal protein L32: 747 C30C11.1 orthologpMON101055 Dv232_B0250_1; putative rpl-2, structural constituent ofribosome: 748 B0250_1 ortholog pMON98446 Dv233_F54C9_5; putative rpl-5,5S rRNA binding, structural 749 constituent of ribosome: F54C9_5ortholog pMON101138 Dv235_C04F12.4; putative rpl-14, large ribosomalsubunit L14 protein: 750 C04F12.4 ortholog pMON98449 Dv236_C01G8_5;putative erm-1, Ezrin/Radixin/Moesin (ERM) family 751 of cytoskeletallinkers: C01G8_5 ortholog pMON98439 Dv237_F57B9_10; putative rpn-6,proteasome Regulatory Particle, 752 Non-ATPase-like: F57B9_10 orthologpMON98436 Dv240_F53A3_3; putative rps-22, structural constituent ofribosome: 753 F53A3_3 ortholog pMON101078 Dv241_F32H2.5; putativealcohol dehydrogenase, zinc-dependent: 754 F32H2.5 ortholog pMON101058Dv242_B0336_2; putative arf-1, small monomeric GTPase: B0336_2 755ortholog pMON101057 Dv244_C14B9_7; putative rpl-21, structuralconstituent of ribosome: 756 C14B9_7 ortholog pMON98444 Dv245_C26F1_4;putative rps-30, Ribosomal Protein, Small subunit: 757 C26F1_4 orthologpMON98434 Dv247_C13B9_3; putative delta subunit of the coatomer (COPI)758 complex: C13B9_3 ortholog pMON101053 Dv248_F38E11_5; putativevesicle coat complex COPI, beta' subunit: 759 F38E11_5 orthologpMON98445 Dv249_F37C12_9; putative rps-14, structural constituent ofribosome: 760 F37C12_9 ortholog pMON101056 Dv250_CD4_6; putative pas-6,endopeptidase: CD4_6 ortholog 761 pMON101104 Dv251_D1007.12; putativerpl-24.1, structural constituent of ribosome: 762 D1007.12 orthologpMON101088 Dv252_C49H3.11; putative rps-2, structural constituent ofribosome: 763 C49H3.11 ortholog pMON101079 Dv253_C26D10.2; putativehel-1, ATP-dependent RNA helicase: 764 C26D10.2 ortholog pMON101085Dv254_B0336.10; putative rpl-23, structural constituent of ribosome: 765B0336.10 ortholog pMON38879 Dv255_C36A4.2; putative member of CytochromeP450 family: 766 C36A4.2 ortholog pMON101087 Dv256_K05C4.1; putativepbs-5, proteasome beta subunit: K05C4.1 767 ortholog pMON101082Dv257_F29G9.5; putative rpt-2, 26S proteasome regulatory complex: 768F29G9.5 ortholog pMON101084 Dv258_F40F8.10; putative rps-9, structuralconstituent of ribosome: 769 F40F8.10 ortholog pMON101083 Dv259_K07D4.3;putative rpn-11, 26S proteasome regulatory complex, 770 subunit RPN11:K07D4.3 ortholog pMON101080 Dv260_F49C12.8; putative rpn-7, proteasomeRegulatory Particle, Non- 771 ATPase-like: F49C12.8 ortholog pMON101115Dv261_D1054.2; putative pas-2, endopeptidase: D1054.2 ortholog 772pMON101141 Dv263_F55A3.3; putative metalloexopeptidase: F55A3.3 ortholog773 pMON101126 Dv264_F56F3.5; putative rps-1, structural constituent ofribosome: 774 F56F3.5 ortholog pMON101133 Dv266_C09D4.5; putativerpl-19, structural constituent of ribosome: 775 C09D4.5 orthologpMON38881 Dv268_R06A4.9; putative Polyadenylation factor I complex,subunit 776 PFS2: R06A4.9 ortholog pMON101135 Dv271_F37C12.4; putativerpl-36, structural constituent of 777 ribosome: F37C12.4 orthologpMON101132 Dv273_F54E7.2; putative rps-12, structural constituent ofribosome: 778 F54E7.2 ortholog pMON101139 Dv274_C23G10.4; putativerpn-2, proteasome Regulatory Particle, 779 Non-ATPase-like: C23G10.4ortholog pMON101130 Dv275_C03D6.8; putative rpl-24.2, structuralconstituent of ribosome: 780 C03D6.8 ortholog pMON101119 Dv276_C26E6.4;putative DNA-directed RNA polymerase: C26E6.4 781 ortholog pMON101134Dv277_R13A5.8; putative rpl-9, structural constituent of ribosome: 782R13A5.8 ortholog pMON101127 Dv279_F42C5.8; putative rps-8, structuralconstituent of ribosome: 783 F42C5.8 ortholog pMON101122 Dv280_F13B10.2;putative rpl-3, large ribosomal subunit L3: F13B10.2 784 orthologpMON101116 Dv281_T05C12.7; putative cct-1, Chaperonin complex component,785 TCP-1 alpha subunit: T05C12.7 ortholog pMON101125 Dv282_F07D10.1;putative rpl-11.2, structural constituent of ribosome: 786 F07D10.1ortholog pMON38883 Dv283_T05H4.6; putative Peptide chain release factor1 (eRF1): 787 Dv283_T05H4.6 ortholog pMON101124 Dv284_C47E8.5; putativedaf-21, Heat shock 90 protein, chaperone 788 activity: C47E8.5 orthologpMON101120 Dv285_M03F4.2; putative act-4, actin: M03F4.2 ortholog 789pMON101137 Dv286_F25H5.4; putative eft-2, translation elongation factor:F25H5.4 790 ortholog pMON101140 Dv287_F26D10.3; putative hsp-1, Heatshock protein: F26D10.3 791 ortholog pMON101117 Dv288_F28D1.7; putativerps-23, structural constituent of ribosome: 792 F28D1.7 orthologpMON38886 Dv290_CG11979; putative H-exporting ATPase: CG11979 ortholog793 pMON38885 Dv291_CG13628; putative H-exporting ATPase: CG13628ortholog 794 pMON101103 Dv293_CG31237; putative DNA-directed RNApolymerase II: CG31237 795 ortholog pMON101096 Dv294_CG8669; putativecryptocephal; transcription factor, involved in 796 molting cycle,pupariation and metamorphosis: CG8669 ortholog pMON101095 Dv295_CG8048;putative Vacuolar H+ ATPase 44 kD C subunit: 797 CG8048 orthologpMON101100 Dv298_CG9032; putative H-exporting ATPase: CG9032 ortholog798 pMON101111 Dv299_CG17369; putative H-exporting ATPase: CG17369ortholog 799 pMON101129 Dv303_CG4152; putative ATP-dependent RNAhelicase: CG4152 800 ortholog pMON101136 Dv305_CG4916; putativeATP-dependent RNA helicase: CG4919 801 ortholog pMON101131 Dv315_CG9160;putative NADH dehydrogenase: CG9160 ortholog 802 pMON101123Dv316_CG8764; putative ubiquinol-cytochrome-c reductase: CG8764 803ortholog pMON98364 C4_Dv49_CG8055 concatemer; putative binding, carrieractivity: 804 CG8055 ortholog pMON98365 C5_Dv49_CG8055 concatemer;putative binding, carrier activity: 805 CG8055 ortholog. pMON98368 C6concatemer of highly effective WCR targets, ~50% GC criterion, 806consisting of segments in order 5′-3′ from Dv26, Dv49, Dv23, Dv20, Dv13,Dv22, Dv18 pMON98369 C7 concatemer of insect-specific targets, ~50% GCcriterion, consisting 807 of segments in order 5′-3′ from Dv6, Dv1,Dv88, Dv93, Dv4, Dv113, Dv127, Dv99 pMON98372 C8 concatemer; putativesodium/potassium-exchanging ATPase: 808 CG9261 ortholog pMON98373 C9concatemer; putative sodium/potassium-exchanging ATPase: 809 CG9261ortholog pMON98366 C10 concatemer of genes with putative same/differentmode of action, 810 ~50% GC criterion pMON98367 C12 concatemer of highlyeffective WCR targets, ~50% GC criterion, 811 consisting of segments inorder 5′-3′ from Dv23-Dv13-Dv26-Dv18- Dv49-Dv22-Dv20. pMON98371 C14concatemer of gene targets active in several different organisms, 812~50% GC criterion pMON98503 Comprising DV49 putative ESCRT-III(endosomal sorting complex 820 required for transport III) complexsubunit from Diabrotica virgifera pMON98504 Comprising putative Vha68-2:CG3762 ortholog; 250 bp concatamer C1; 821 pMON102862 Dv319_CG14750;putative ESCRTII, Vps25: CG14750 ortholog 835 pMON102863 Dv320_CG9712;putative ESCRTI, Vps23: CG9712 ortholog 836 pMON102861 Dv321_CG12770;putative ESCRTI, Vps28: CG12770 ortholog 837 pMON102865 Dv322_CG14542;putative ESCRT III, Vps2: CG14542 ortholog 838 pMON102866 Dv323_CG4071;putative ESCRT III, Vsp20: CG4071 ortholog 839 pMON102871 Dv326_CG3564;putative protein carrier, component of the COPI vesicle 840 coat: CG3564ortholog pMON102873 Dv327_CG6223; putative coatomer, component of theCOPI vesicle 841 coat: CG6223 ortholog pMON102877 Dv328_CG6948; putativeClathrin light chain, coat of coated pit: 842 CG6948 ortholog pMON102872Dv330_CG9543; putative COPI vesicle coat: CG9543 ortholog 843 pMON102879Dv331_CG5183; putative KDEL sequence binding: CG5183 ortholog 844pMON102867 Dv335_F11C1.6; putative nhr-25, DNA binding: F11C1.6 ortholog845 pMON102870 Dv337_CG18734; putative furin 2, serine-typeendopeptidase: CG18734 846 ortholog pMON102875 Dv329_CG7961; putativecoatomer, component of the COPI vesicle 874 coat: CG7961 ortholog

Efficacy tests were conducted as follows, utilizing progeny of cornplants transformed with selected insect control constructs:

1. Seven days post planting: incubate 10,000 WCR eggs per event (10plants per event) at 25° C., 60% RH in complete darkness for seven days.

2. Fourteen days post infestation: plants are transplanted from 4″ peatpots to 8″ pots; v4 root tip samples may be taken for gene expressionstudies.

3. Fourteen days post planting: wash the WCR eggs out of the soil. Placethe eggs and soil into a 60-mesh screen and place a 30-mesh screen ontop of the 60-mesh screen to protect the eggs from the water stream.Rinse thoroughly with warm water using a spray nozzle until the soil isremoved.

4. Suspend the eggs in a 2% (w/v) Difco agar solution, 25 mL of solutionper 1 mL of eggs. The eggs are infested into the soil in about 3 or 4aliquots using, for example, an Eppendorf repeater pipette, about 1000eggs per plant. Holes are made into the soil using a spatula prior toinfestation and covered after infestation.

5. Twenty-eight days post planting v8 root tip samples may be taken forgene expression studies.

6. Thirty-five days post planting; the assay is evaluated. Plants arecut down using pruning sheers, leaving about 6″ of stalk. Plant stakescontaining the event information are hole-punched and zip tied to thestalk. As much soil as possible is removed from the root system. Theremainder of the soil is washed off using a spray hose.

7. The roots are examined and given a root damage rating by using theOlsen (0-3) NIS scale for WCR larvae damage.

FIG. 1 and FIG. 2 illustrate insect control results obtained followingchallenge of F1 corn plants (derived from plants transformed with eitherpMON98503 or pMON98504) with WCR. In growth chamber efficacy testsperformed essentially as described above, NIS scores at or below theeconomic injury threshold were seen in progeny of events derived bytransformation with pMON98503 or pMON98504.

Full length EST DNA sequences were assembled for selected genesdescribed in Examples 3 and 7 displaying significant activity versusWestern Corn Rootworm. These EST sequences are listed in Table 7:

TABLE 7 Assembled EST Sequences for WCR Targets Assembled Sequence SEQID NO Dv9 full length (aka Apple); putative ortholog of CG2331 813 Dv10full length (Rp19); putative ortholog of CG6141 814 Dv11 full length(Rp119); putative ortholog of CG2746 815 Dv13 full length (Rps4);putative ortholog of CG11276 816 Dv35 full length v-ATPase A; orthologof CG3762 817 Dv49 full length ortholog of CG8055 818 Dv248 full lengthputative ortholog of CG6699 819

Example 8 Creation and Efficacy Results for Dv49 and Dv248 Sequences

Portions of the assembled EST and adjacent sequences of Dv49 (SEQ IDNO:818) and Dv248 (SEQ ID NO:819) were selected for further bioactivityassays based on criteria including predicted function, phenotype ofknockout mutants of corresponding coding regions in other organisms,segment length, GC content, similarity to known sequences, and predictedsecondary structure (e.g. Elbashir, et al., 2001b). The respective Dv49and Dv248 sequences were synthesized in vitro based on their predictedactivity against WCR individually, as well as grouped as shown in FIGS.3-4, and Table 8, and applied to WCR larvae.

TABLE 8 Dv49 and Dv248 Fragments Assessed for Efficacy Against WCRFragment SEQ ID NO F1 822 F2 823 F3 824 F4 825 F5 826 F6 827 F7 828 F8829 F9 830 F10 831 F11 832 F12 833 F13 834

Fragments F1-F3 correspond to portions of the full length Dv49transcript. Fragments F4-F6 correspond to portions of the Dv248transcript or flanking region. Fragments F7-F13 are concatemers of twoor more of fragments F1-F6, as shown in FIG. 4. Fragment F13 (SEQ IDNO:834) represents the C38 (Dv49-Dv248) concatemer.

Dose Response data for F1-F13 is shown in FIGS. 5-7. As shown in FIG. 5,activity (larval % mortality) of fragments F4-F6, comprisingDv248-derived sequences (FIG. 4), was significantly better than thecontrol when fed to WCR larvae at 0.1 ppm. Fragment F6 displayedsignificant activity when fed at 0.02 ppm as well. Activity of fragmentF5 at the lowest dose is likely an artifact, since surviving larvaedisplayed no stunting.

As shown in FIG. 6, each of fragments F7-F10 displays statisticallysignificant activity when fed to WCR larvae at 0.1 ppm. Fragments F9 andF10 also displayed statistically significant activity when fed to WCRlarvae at 0.02 ppm, and fragment F10 displayed statistically significantactivity when fed to WCR larvae at 0.01 ppm as well. Activity of F8 at0.01 ppm may be an artifact.

As shown in FIG. 7, fragments F11-F13 display statistically significantactivity when fed to WCR larvae at 0.02 ppm or higher. Additionally, thelargest fragments (F12 and F13) show activity at 0.005 ppm and higher.

Example 9 Additional Active Concatemer Sequences Derived From ConcatemerC6

As shown in Table 9, portions of active concatemer C6 (SEQ ID NO:806),derived from pMON98368, were identified in diet overlay bioassays(performed as described e.g. in Example 2) as inhibiting the growthand/or survival of corn rootworm (WCR). The C6 full length concatemercontains 7 target subfragments of 70 bp each, as noted in Table 6 andTable 9.

TABLE 9 Efficacy of full length C6 concatemer and selected sub-portionsin rootworm diet bioassay (SEQ ID NOs: 806, 847-873). SEQ ID ConcatemerSegments % Mortality at 1 ppm NO C6_full lengthDv26-Dv49-Dv23-Dv20-Dv13-Dv22- 100 806 Dv18 C6.1 Dv26-Dv49 35.3 847 C6.2Dv26-Dv49-Dv23 57.1 848 C6.3 Dv26-Dv49-Dv23-Dv20 84.7 849 C6.4Dv26-Dv49-Dv23-Dv20-Dv13 51 850 C6.5 Dv26-Dv49-Dv23-Dv20-Dv13-Dv22 41.3851 C6.6 Dv49-Dv23 94.6 852 C6.7 Dv49-Dv23-Dv20 75.6 853 C6.8Dv49-Dv23-Dv20-Dv13 50 854 C6.9 Dv49-Dv23-Dv20-Dv13-Dv22 56.4 855 C6.10Dv49-Dv23-Dv20-Dv13-Dv22-Dv18 52.5 856 C6.11 Dv23-Dv20 74.6 857 C6.12Dv23-Dv20-Dv13 63.4 858 C6.13 Dv23-Dv20-Dv13-Dv22 58.5 859 C6.14Dv23-Dv20-Dv13-Dv22-Dv18 60.8 860 C6.15 Dv20-Dv13 57.5 861 C6.16Dv20-Dv13-Dv22 20 862 C6.17 Dv20-Dv13-Dv22-Dv18 44.8 863 C6.18 Dv13-Dv2271.1 864 C6.19 Dv13-Dv22-Dv18 52.5 865 C6.20 Dv22-Dv18 44.6 866 C6.28Dv26 58.9 867 C6.29 Dv49 72.3 868 C6.30 Dv23 71.9 869 C6.31 Dv20 62.6870 C6.32 Dv13 54.2 871 C6.33 Dv22 56.7 872 C6.34 Dv18 44.6 873

Full length concatemer C7 contains 8 target sub-fragments of 70 bp each,as noted in Table 6. Similarly, full length concatemer C12 contains 7target sub-fragments from Dv23-Dv13-Dv26-Dv18-Dv49-Dv22-Dv20, ranging inlength between 53-80 bp, as noted in Table 6 and Table 10.

TABLE 10 Composition of full length C12 concatemer and selectedsub-portions (SEQ ID NOs: 811, 887-892). Included sequence (SEQ ID NO)in 5′-3′ Concatemer Segments direction C12_fullDv23-Dv13-Dv26-Dv18-Dv49-Dv22-Dv20 811 length C12.1 Dv23-Dv13 887 C12.2Dv23-Dv13-Dv26 888 C12.3 Dv23-Dv13-Dv26-Dv18 889 C12.4Dv23-Dv13-Dv26-Dv18-Dv49 890 C12.5 Dv23-Dv13-Dv26-Dv18-Dv49-Dv22 891C12.6 Dv13-Dv26 892 C12.7 Dv13-Dv26-Dv18 893 C12.8 Dv13-Dv26-Dv18-Dv49894 C12.9 Dv13-Dv26-Dv18-Dv49-Dv22 895 C12.10Dv13-Dv26-Dv18-Dv49-Dv22-Dv20 896 C12.11 Dv26-Dv18 897 C12.12Dv26-Dv18-Dv49 898 C12.13 Dv26-Dv18-Dv49-Dv22 899 C12.14Dv26-Dv18-Dv49-Dv22-Dv20 900 C12.15 Dv18-Dv49 901 C12.16 Dv18-Dv49-Dv22902 C12.17 Dv18-Dv49-Dv22-Dv20 903 C12.18 Dv49-Dv22 904 C12.19Dv49-Dv22-Dv20 905 C12.20 Dv22-Dv20 906

Table 11 lists additional sequences for use in targeting othercoleopteran pests, including Diabrotica sp. and other Coccinellidae andChrysomelidae.

TABLE 11 Additional Coleopteran Target Sequences Sequence and source SEQID NO Dbar248_CG6699 (D. barberi) 875 Dbal248_CG6699 (D. balteata) 876Du248_CG6699 (D. undecimpunctata howardi) 877 Dz248_CG6699 (D. virgiferazea) 878 Dv248_CG6699 (D. virgifera virgifera) 879 Ev248_CG6699(Epilachna varivestis) 880 Ld248_CG6699 (Leptinotarsa decemlineata) 881Dbal49_CG8055_2 (D. balteata) 882 Db49_CG8055_2 (D. barberi) 883Du49_CG8055_2 (D. undecimpunctata howardi) 884 Dz49_CG8055_2 (D.virgifera zea) 885 Dv49_CG8055_2 (D. virgifera virgifera) 886

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent they provide exemplaryprocedural or other details supplemental to those set forth herein, arespecifically incorporated herein by reference:

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1. A method for controlling coleopteran pest infestation comprising:providing in a diet of a coleopteran pest an agent comprising a firstpolynucleotide sequence and a second polynucleotide sequence whereinsaid first polynucleotide sequence exhibits from about 95 to about 100%nucleotide sequence identity along at least from about 19 to about 25contiguous nucleotides to a coding sequence according to SEQ ID NO:818and is hybridized to said second polynucleotide sequence that iscomplementary to said first polynucleotide sequence, and; wherein saidagent functions upon ingestion by the pest to inhibit a biologicalfunction within said pest thereby controlling pest infestation.
 2. Themethod of claim 1, wherein the first and second polynucleotide sequencesare expressed in a plant cell to produce a double stranded ribonucleicacid that functions upon ingestion by the pest to inhibit the expressionof a target sequence within said pest.
 3. The method of claim 1, whereinthe pest exhibits reduced growth following said ingestion.
 4. The methodof claim 2, wherein the plant cell further comprises a polynucleotidesequence encoding a pesticidal agent selected from the group consistingof a patatin, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporus insecticidal protein, and a Bacillus sphaericusinsecticidal protein.
 5. The method of claim 4 wherein said Bacillusthuringiensis insecticidal protein is selected from the group consistingof a Cry1, a Cry3, a TIC851, a CryET70, a Cry22, a TIC901, a TIC201, aTIC407, a TIC417, a binary insecticidal protein CryET33 and CryET34, abinary insecticidal protein CryET80 and CryET76, a binary insecticidalprotein TIC100 and TIC101, a combination of an insecticidal protein ET29or ET37 with an insecticidal protein TIC810 or TIC812, and a binaryinsecticidal protein PS149B1.
 6. The method of claim 1, wherein saidcoleopteran pest is a Diabrotica spp. selected from the group consistingof Diabrotica virgifera, Diabrotica virgifera virgifera, Diabroticavirgifera zea, Diabrotica balteata, Diabrotica barberi, Diabroticaviridula, Diabrotica speciosa, and Diabrotica undecimpunctata.
 7. Themethod of claim 1, wherein the pest exhibits reduced feeding followingsaid ingestion.
 8. The method of claim 2, wherein the plant cell is acorn plant cell.
 9. The method of claim 2, wherein the first and secondpolynucleotide sequences are expressed from a construct integrated inthe genome of the plant cell.
 10. The method of claim 2, wherein thefirst and second polynucleotide sequences are expressed as a singlepolynucleotide molecule.
 11. The method of claim 10, wherein the firstand second polynucleotide sequences are expressed as a singlepolynucleotide molecule that forms a stem-loop structure uponexpression.